Specific antibodies to kringle 5 of apo(a) and methods of use therefor

ABSTRACT

The present invention provides monoclonal antibodies specific for kringle 5 of apo(a) and hybridomas secreting such antibodies. The invention also relates to assay methods for directly measuring concentrations of lipoprotein(a) [Lp(a)] in a plasma sample. In one embodiment, the method involves the specific capture of Lp(a) from a plasma sample with a monoclonal antibody developed against kringle 5 of apo(a), which is non-cross-reactive with plasminogen and kringle 4 of apo(a). The quantity of the Lp(a) present in the sample is then measured by detecting the amount of Lp(a)-anti-kringle 5 complex that has formed in the reaction. Alternatively, the Lp(a) may be captured non-specifically and then detected with the monoclonal antibody specific for kringle 5 of apo(a). The invention also provides competitive assays using the above-mentioned kringle 5 specific monoclonal antibodies.

RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) toprovisional application U.S. Ser. No. 60/072,924, filed Jan. 20, 1998,the disclosure of which is incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates generally to antibodies whichspecifically bind to kringle 5 of apo(a), to hybridoma cell lines whichsecrete those antibodies, to methods of using the antibodies and to kitsfor measuring lipoprotein (a) from plasma.

BACKGROUND OF THE INVENTION

[0003] Lipoprotein(a) [Lp(a)] was described as a genetic variant of lowdensity lipoprotein (LDL) in 1963 (K. Berg (1963) Acta Pathol MicrobiolScand 59: 369-381). Later it was discovered that although Lp(a)resembles LDL in having similar lipid composition and a commonapolipoprotein B-100 (apo B), Lp(a) contains an additional glycoprotein,named apolipoprotein(a) [apo(a)]. Each Lp(a) molecule contains onemolecule of apo(a) per one molecule of apo B covalently linked by asulfide bond that can be easily reduced to LDL and apo(a) (Gaubatz etal. (1983) J. Biol Chem 258: 4582-4589; Fless et al. (1984) J. Biol Chem259: 11470-11478; Fless et al. (1986) J Biol Chem 261: 8712-8718, Flesset al (1994) Biochemistry 33: 13492-13501; Marcovina and Morrisett(1995) Curr Opin in Lipidology 6: 136-145; Albers et al. (1996) J LipidRes 37: 192-196).

[0004] Lipoprotein(a) particles exhibit considerable inter- andintra-individual heterogeneity, with some individuals exhibiting two ormore distinct Lp(a) particles differing in hydrated density (Fless etal. (1984) J Biol Chem 259: 11470-11478). Also, the Lp(a) particlevaries widely in size, with the size heterogeneity related primarily tothe size of the apo(a) isoforms, ranging from 280 to 838 KDa; to date,34 different isoforms have been identified (Marcovina et al. (1993)Biochem Biophys Res Commun 191: 1192-1196). The number of apo(a)isoforms that can be distinguished varies from six to at least twelveisoforms. The smaller isoforms are generally present at less frequencyand are associated with the higher Lp(a) concentrations, whereas thelarger isoforms have a higher frequency and are associated with lowerLp(a) concentrations. There appears to be an inverse relationshipbetween the apparent molecular mass of the apo(a) isoforms and theconcentrations of Lp(a) in plasma (G. Utermann (1989) Science 246:904-910; Morrisett et al. (1990) in Lipoprotein(a), Academic Press. pp.53-74; Sandholzer et al. (1992) Arteriosclerosis and Thrombosis 12:1212-1226).

[0005] The structural gene for apo(a) is located on chromosome 6 nearthe plasminogen gene (Frank et al. (1988) Hum Genet 79: 352-356).Sequencing of apo(a) at both the protein and cDNA level has revealed ahigh degree of homology to plasminogen (Eaton et al. (1987) Proc NatlAcad Sci 84: 3224-3228; McLean et al. (1987) Nature (London) 330:132-137). Apo(a) contains two types of plasminogen-like domains: asingle kringle 5 domain, with 82% amino acid sequence homology and 91%nucleotide sequence homology with plasminogen, and multiple repeats of akringle 4 domain, with 61-75% amino acid homology and 75-85% nucleotidesequence homology with the kringle 4 domain of plasminogen. Homology toplasminogen is also revealed by immunochemical studies that showcross-reactivity of apo(a) and plasminogen (Karadi et al. (1988) BiochimBiophys Acta 960: 91-97); Lafferty et al. (1991) J Lipid Res 32:277-292).

[0006] Numerous studies have indicated that elevated levels of Lp(a) inplasma are associated with premature coronary heart disease (CHD) (Scanuand Fless (1990) J Clin Invest 85: 1709-1715; Sandholzer et al. (1992)Arteriosclerosis and Thrombosis 12: 1214-1226, Seed et al. (1990) NewEngl J Med 332: 1494-1499; (Genest et al. (1992) J Am Coll Cardiol 19:792-802; Dahlen et al. (1986) Circulation 74: 758-765). Lp(a)concentrations in human plasma range from 1 mg/dL to more than 100mg/dL. When the plasma Lp(a) level is above 30 mg/dL, the relative riskof CHD is raised about two-fold. When LDL and Lp(a) are both elevated,the relative risk is increased to about five-fold (Armstrong et al.(1986) Atherosclerosis 62: 249-257). Recent studies have suggested thatincreased Lp(a) concentrations may inhibit fibrinolysis by reducing thegeneration of plasmin by competing for plasminogen cell-surfacereceptors, or inhibiting activation of plasminogen, or competing forbinding sites on fibrin (Hajjar et al. (1989) Nature (London) 339:303-305; Miles et al. (1989) Nature (London) 339: 301-303;Gonzalez-Gronow et al. (1989) Biochemistry 28: 2374-2377; Edelberg etal. (1989) Biochemistry 28: 2370-2374; Loscalzo et al. (1990)Anteriosclerosis 10: 240-245; Harpel et al. (1989) Proc Natl Acad SciUSA 86: 3847-3851; Angles-Cano (1994) Chem Phys Lipids 67/68: 353-362;369-380; Liu et al. (1994) Biochemistry 33: 2554-2560; Hajjar andNachman (1996) Annu Rev Med 47: 423-442).

[0007] More recently, it has been shown that the binding activity of themacrophage Lp(a)/apo(a) receptor can be blocked by a monoclonal antibodydirected against a specific kringle 4 domain (subtypes 6-7) (Keesler etal (1996) J Biol Chem 27: 32096-32104). This suggests a possible role ofLp(a) in Lp(a)-induced atherogenesis. While the function of Lp(a) isunknown, a significant correlation has been established between elevatedlevels of Lp(a) and coronary artery and cardiovascular disease that ledmany scientists to study the physiological role of Lp(a) in heartdisease (R. M. Lawn (1992) Scientific American pp. 54-60; Simon et al.(1993) Curr Opin in Lipidology 8: 814-820; Klezovitz and Scanu (1995)Curr Opin in Lipidology 6: 223-228; Durrington (1995) Bailliere ClinEndocrinol 9: 773-795).

[0008] A number of assay methods for quantitating Lp(a) in plasma areknown (see Morrisett et al. (1987) in Plasma Lipoproteins, ElsevierScience B.V., Chapter 5, pp. 129-152; (Gaubatz et al. (1986) in Methodsin Enzymology, Vol. 129, pp. 167-187; Albers et al. (1990) Clin Chem 36:2019-2026; Labeur and Rosseneu (1992) Curr Opin in Lipidology 3:372-376; Albers and Marcovina (1994) Curr Opin in Lipidology 5:417-421). The assays include radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISAs), radial immunodiffusion,electroimmunoassays, immunoelectrophoresis and turbidimetric assays.Most of the Lp(a) assay methods except the ELISAs are not commonly useddue to inherent technical problems (Labeur and Rosseneu (1992) Curr Opinin Lipidology 3: 372-376). ELISAs that are presently known use eithermonoclonal or affinity-purified polyclonal antibodies. The majority ofthe monoclonal antibodies recognize the kringle 4 epitope of apo(a),whereas the polyclonal antibodies recognize both kringle 4 and kringle 5epitopes of apo(a) (Lafferty et al. (1991) J Lipid Res 32: 277-292;Fless et al. (1989) J Lipid Res 30: 651-662; Rainwater and Manis (1988)Atherosclerosis 73: 23-31).

[0009] As noted above, apo(a) contains multiple copies of kringle 4domain. The multiple copies of apo(a) kringle 4 are similar but notidentical to each other and can be divided into 10 distinct kringletypes (kringle 4 types 1 through 10). One copy each of kringle 4 type 1and types 3 through 10 is present per apo(a) molecule; kringle 4 type 2,however, is present in a variable number of repeats (from 3 to >40) andare therefore responsible for the size heterogeneity of apo(a) andconsequently Lp(a) (Lackner et al. Hum Molec Genet (1993) 2: 933-940;Van der Hoek et al. Hum Molec Genet (1993) 2: 361-366). From thestructural sequence of kringle 4 repeats it seems obvious that theimmunoreactivity of the antibodies used in the immunoassays to measureLp(a) concentrations will vary according to the number of epitopesavailable in a particular Lp(a). Therefore, antibodies against apo(a)should be selected to be specific for that part of the apo(a) moleculethat is independent of size polymorphism, i.e. for kringle 4 domainsother than type 2 or kringle 5 domain.

[0010] Among the numerous papers published to date, only one reports thedomain specificity of the monoclonal antibodies used in the immunoassaysto measure Lp(a) (Marcovina et al. (1995) Clin. Chem 41: 246-255).Recently, an immunoassay method for the detection of Lp(a) was disclosedusing an anti-apo(a) monoclonal antibody that was described asnon-reactive with plasminogen and the kringle 4 type 2 repeats of apo(A)(see WO96/19500 published Jun. 27, 1996). Although Albers, Rosseneau,and others have suggested that an optimal antibody should be the onethat is directed towards an epitope that is localized in thenon-repetitive and non-glycosylated kringle 5 domain (Albers et al.(1990) Clin Chem 36: 2019-2026; Labeur and Rosenau (1992) Curr Opin inLipidology 3: 372-376; Albers and Marcovina (1994) (Curr Opin inLipidology 5: 417-421), it was not been possible until recently todevelop kringle 5 domain specific antibodies because of extensiveproblems associated with generating domain specific antibodies.

[0011] A polyclonal antibody was recently developed by immunizing asheep with a cloned kringle 5 fusion protein (Chenivesse et al. (1996)Protein Expression and Purification 8: 145-150). This antibody was shownby ELISA and Western blot to react with Lp(a) and the C-terminal domainof apo(a), but not with the kringle 4 repeats at the N-terminal end. Inboth formats, the proteins were immobilized on solid phases, sometimesunder denaturing conditions. No data was provided on whether thispolyclonal antibody cross-reacted with plasminogen or any of the otherlipoproteins that are abundant in human plasma.

[0012] The reactivity of an antibody for its specific antigen can differconsiderably depending on the type of assay format it is used in, i.e.how and where in the assay the antibody is utilized. The state of theantigen, e.g. whether it is in solution or attached to a solid phase,how it is attached to a solid phase, whether it is denatured or not,also affects antibody binding; some antibodies recognizeconformation-dependent epitopes and therefore require the antigen to bein its native state. Moreover, the specificity and immunoreactivity ofpolyclonal antibodies can vary from animal to animal and species tospecies making it difficult to produce a reliable and consistentimmunoassay. Therefore, monoclonal antibodies are presently needed whichare specific for an epitope(s) that are localized in the kringle 5domain of apo(a) and do not cross-react with plasminogen or the kringle4 domain of apo(a). Such monoclonal antibodies may serve as accuratemarkers for the detection and diagnosis of heart disease.

SUMMARY OF THE INVENTION

[0013] One object of the present invention is provide highly specificmonoclonal antibodies against the kringle 5 domain of apo(a). Anotherobject of the invention is to develop an assay for Lp(a) that is notaffected by the variability in size, structure and difference inglycosylation of the kringle 4 repeats of apo(a).

[0014] The present invention relates to methods for determining theamount of Lp(a) in a test sample. In one embodiment, the methodcomprises the steps of (a) contacting the sample and an Lp(a) specificbinding agent coupled to a solid support wherein the Lp(a) specificbinding agent is a monoclonal antibody or fragment thereof thatspecifically binds to kringle 5 of apo(a) for a time and underconditions to form binding agent-Lp(a) complexes; and (b) determiningthe amount of Lp(a) bound to the binding agent-Lp(a) complexes. In apreferred embodiment, the Lp(a) binding agent is a monoclonal antibodyor fragment thereof which binds to substantially all Lp(a) via kringle 5of apo(a), to plasminogen at less than 1% of Lp(a) binding and to LDL,VLDL, IDL and HDL at less than 2% of Lp(a) binding. In a more preferredembodiment, the solid support is separated from the sample beforedetermining the amount of Lp(a) bound to the complexes. The solidsupport may be selected from the group consisting of nitrocellulose,latex, nylon, polystyrene, beads, particles, magnetic particles, andglass fiber. In a most preferred embodiment, the monoclonal antibody isselected from the group consisting of 1-532-266, 1-390-191, 1-458-165,1-892-230, 1-292-189, 1-431-378, 1-746-183, and 1-546-264.

[0015] In an alternative embodiment, the method further comprisescontacting an indicator reagent to the sample and Lp(a) specific bindingagent prior to step (b) above and includes the aforementioned preferredembodiments. In a preferred aspect of this embodiment, the indicatorreagent is selected from the group consisting of K4 specific monoclonalantibody. K4 polyclonal antibody, K4/K5 monoclonal antibody, K4/K5polyclonal antibody and fragments thereof.

[0016] In yet another embodiment, the invention provides a method fordetermining the amount of Lp(a) in a test sample comprising the steps of(a) contacting the sample, a capture reagent bound to a solid support,and an indicator reagent wherein the indicator reagent is a labeledmonoclonal antibody or fragment thereof that specifically binds tokringle 5 of apo(a) for a time and under conditions to form capturereagent-Lp(a)-indicator reagent complexes; and (b) determining theamount of Lp(a) bound to the binding agent-Lp(a)-indicator reagentcomplexes. Alternatively, the indicator reagent is a labeled monoclonalantibody that binds to substantially all Lp(a) via kringle 5, toplasminogen at less than 1% of Lp(a) binding and to LDL, VLDL, IDL andHDL at less than 2% of Lp(a) binding. In this case, as in thosementioned above, a preferred embodiment is one which further comprisesthe step of separating the solid support from the sample beforedetermining the amount of Lp(a) bound to the solid support. Preferablyhere, the capture reagent is selected from the group consisting of K4specific monoclonal antibody, K4 polyclonal antibody, K4/K5 monoclonalantibody. K4/K5polyclonal antibody and fragments thereof. Also,preferably, the indicator reagent is selected from the group consistingof 1-532-266, 1-390-191, 1-458-165, 1-892-230, 1-292-189, 1-431-378,1-746-183, and 1-546-264.

[0017] In yet another embodiment, the invention provides a method fordetermining the amount of Lp(a) in a test sample comprising the steps of(a) contacting the sample, an Lp(a) specific binding agent wherein theLp(a) specific binding agent is conjugated to a first charged substanceand an indicator reagent wherein the indicator reagent is monoclonalantibody or fragment thereof that specifically binds to kringle 5 ofapo(a) for a time and under conditions to form bindingagent-Lp(a)-indicator reagent complexes; (b) contacting the bindingagent-Lp(a)-indicator reagent complexes with an insoluble solid phasematerial which is oppositely charged with respect to the first chargedsubstance, such that the solid phase material attracts and attaches tothe first charged substance; and (c) determining the amount of Lp(a)bound to the binding agent-Lp(a)-indicator reagent complexes.Preferably, the first charged substance is an anionic or cationicmonomer or polymer. More preferably, the indicator reagent is a labeledmonoclonal antibody that binds to substantially all Lp(a) via kringle 5,to plasminogen at less than 1% of Lp(a) binding and to LDL, VLDL, IDLand HDL at less than 2% of Lp(a) binding. Even more preferably, themonoclonal antibody is selected from the group consisting of 1-532-266,1-390-191, 1-458-165, 1-892-230, 1-292-189, 1-431-378, 1-746-183, and1-546-264.

[0018] In another embodiment, the invention provides a method fordetermining the amount of cholesterol associated with Lp(a) in a testsample comprising the steps of (a) contracting the sample and amonoclonal antibody or fragment thereof that specifically binds tokringle 5 of apo(a) wherein the antibody is coupled to a solid support;(b) separating the solid support from the sample; and (c) determiningthe amount of cholesterol bound to the solid support.

[0019] The invention also includes competitive assays for determiningLp(a) in a test sample. One embodiment provides a method for determiningthe amount of Lp(a) in a test sample comprising the steps of (a)contacting the sample and an indicator reagent wherein the indicatorreagent is a monoclonal antibody or fragment thereof that specificallybinds to kringle 5 of apo(a) with a solid support coated with Lp(a) fora time and under conditions to permit binding of the indicator reagentwith Lp(a) in the test sample and with the Lp(a) bound to the solidsupport; and (b) determining the amount of Lp(a) in the test sample bydetecting the reduction in binding of the indicator reagent to the solidsupport as compared to the signal generated from a negative sample toindicate the presence of Lp(a) in the test sample. In an alternativemethod, the indicator reagent is replaced by labeled Lp(a) or labeledkringle 5 of apo(a) and the bound Lp(a) is replaced by bound monoclonalantibody or a fragment thereof that specifically binds to kringle 5 ofapo(a). In each instance above, a molecule bound to the solid support,whether antigen or antibody, may be bound directly or indirectly. Themonoclonal antibody used in the above-described competitive assayspreferably binds to substantially all Lp(a) via kringle 5, toplasminogen at less than 1% of Lp(a) binding and to LDL, VLDL, IDL andHDL at less than 2% of Lp(a) binding. More preferably, the monoclonalantibody is selected from the group consisting of 1-532-266, 1-390-191,1-458-165, 1-892-230, 1-292-189, 1-431-378, 1-746-183, and 1-546-264.

[0020] The invention further provides a monoclonal antibody specific forLp(a) prepared by a method comprising the steps of (a) immunizing amouse or a rat with kringle 5 of apo(a) or a fragment thereof; (b)making a suspension of mouse or rat spleen cells; (c) fusing the spleencells with mouse or rat myeloma cells in the presence of a fusionpromoter; (d) culturing the fused cells; (e) determining the presence ofanti-Lp(a) antibody in the culture media; (f) cloning a hybridomaproducing antibody that binds to substantially all Lp(a), to plasminogenat less than 1% of Lp(a) binding and to other lipoproteins, such as,LDL, VLDL, IDL and HDL at less than 2% of Lp(a) binding; and (g)obtaining the antibody from the hybridoma.

[0021] The invention also includes a monoclonal antibody specific forLp(a) wherein the antibody binds to (i) substantially all Lp(a) viakringle 5 of apo(a), (ii) plasminogen at less than 1% of Lp(a) bindingand (iii) LDL, VLDL, IDL, and HDL at less than 2% of Lp(a) binding. Theantibody may be an IgG or IgM isotype. A preferred IgG isotype isselected from the group consisting of 1-532-266, 1-390-191, 1-458-165and 1-892-230. A most preferred IgG isotype is 1-892-230. A preferredIgM isotype is selected from the group consisting of 1-292-189,1-431-378, 1-746-183, and 1-546-264. The invention also provideshybridoma cell lines that secrete the above-mentioned monoclonalantibodies.

[0022] The invention further provides a test kit for the detection andquantification of Lp(a) in a plasma sample, comprising a reagent orlabeled reagent which specifically binds to kringle 5 of apo(a).

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIG. 1A is a pictorial representation of apolipoprotein (a). Theapo(a) gene has four structural domains: a signal sequence (SS) which isnearly identical to that of plasminogen, a K4 region which contains12-51 copies of the K4 repeat sequence, a single copy of kringle 5 (K5)and a protease domain. For K4 repeats, the numbers 1 through 10represent the types of K4 structures seen.

[0024]FIG. 1B shows antibody titer plots of monoclonal antibody1-892-230 obtained by incubating microtiter plates coated with Lp(a)(□), Kringle 5 (+), LDL, (⋄), VLDL, (Δ), IDL (×), and HDL (∇) inseparate wells, and measuring the antibody bound to the lipoproteinsusing an ELISA. The antibody concentration (μg/mL) is shown on thex-axis and absorbance at 490 nm on the y-axis.

[0025]FIGS. 2A and 2B are antibody titer plots of four IgG and four IgMmonoclonal antibodies, respectively, obtained by incubating theseantibodies with Lp(a) bound to microtiter plates and measuring theirbinding using an ELISA. In FIG. 2A, monoclonal antibody 1-892-230 isdesignated by the symbol “□”, 1-532-266 by the symbol “+”, 1-390-191 bythe symbol “⋄”, and 1-458-165 by the symbol “Δ”. In FIG. 2B, monoclonalantibody 1-746-183 is designated by the symbol “□”, 1-292-189 by thesymbol “+”, 1-546-264 by the symbol “⋄”, and 1-431-378 by the symbol“Δ”. In each plot, the antibody concentration (μg/mL) is shown on thex-axis and absorbance at 490 nm on the y-axis.

[0026]FIG. 3 shows titration curves of Lp(a) with four IgG monoclonalantibodies obtained by measuring the binding of Lp(a) to microtiterplates which have been coated with the monoclonal antibodies. Themonoclonal antibodies and their symbol designations as well as the x-and y-axis parameters are the same as in FIG. 2A above.

[0027]FIG. 4 shows competitive binding curves of monoclonal antibodies1-892-230 (4A), 1-532-266 (4B) and 1-458-165 (4C) obtained bypre-incubating each antibody with a competitor, adding the mixture tothe microtiter plate to which Lp(a) was already bound, and measuring theantibody bound to the Lp(a) using an ELISA. In FIGS. 4A, 4B, and 4C,Lp(a) is designated by the symbol “□”, kringle 5 by the symbol “+”, andplasminogen by the symbol “⋄”, X- and y-axis parameters are as indicatedabove.

[0028]FIGS. 5A and 5B show the binding curves for the anti-kringle 4monoclonal antibody 4D2 and anti-apo(a) sheep polyclonal antibodiesobtained by incubating these antibodies with microtiter plates coatedwith Lp(a) (□), LDL (+), VLDL (⋄), IDL (Δ), and HDL (×) in separatewells and measuring the antibody bound to the lipoproteins using anELISA. X- and y-axis parameters are as indicated above.

[0029]FIG. 6 shows direct binding curves of the anti-kringle 4monoclonal antibody (Mab) 4D2 and anti-apo(a) sheep polyclonal antibody(Pab) bound to kringle 5 coated on the microtiter plates. In the Figure,monoclonal antibody is designated by the symbol “□” and polyclonalantibody by the symbol “+”. X- and y-axis parameters are as indicatedabove.

[0030]FIGS. 7A and 7B show the binding curves for HRPO-labeledanti-kringle 4 Mab (designated by the symbol “□”) and anti-apo(a)[kringle 4 and kringle 5] Pab (designated by the symbol “+”) to Lp(a)and kringle 5 respectively captured by the anti-kringle 5 Mab 1-892-230bound to microtiter plates. X- and y-axis parameters are as indicatedabove.

[0031]FIG. 8 shows a calibration curve of Lp(a) concentration in mg/dL(x-axis) versus absorbance at 490 nm (y-axis) using anti-kringle 5 asthe capture antibody and HRPO-labeled anti-kringle 4 Mab for detectionas described in Example 4.

[0032]FIGS. 9A and 9B show correlation curves for Lp(a) assays usinganti-kringle 5 Mab 1-358-230 as the capture antibody and HRPO-labeledanti-kringle 4 Mab 4D2 for detection (y-axis) vs. the Terumo ELISA(x-axis). FIG. 9A compares the two assays using normal subjects and FIG.9B uses patients as described in Example 4.

[0033]FIG. 10 shows a calibration curve of Lp(a) concentrationconcentration in mg/dL (x-axis) versus absorbance at 490 nm (y-axis)using anti-kringle 5 Mab as the capture antibody and HRPO-labeledanti-apo(a) Pab for detection as described in Example 5.

[0034]FIGS. 11A and 11B show correlation curves for Lp(a) assays usinganti-kringle 5 Mab 1-892-230 as the capture antibody and HRPO-labeledanti-apo(a) Pab for detection (y-axis) vs. the Terumo ELISA (x-axis).FIG. 11A compares the two assays using normal subjects and FIG. 11B usespatients as described in Example 5.

[0035]FIG. 12 shows a calibration curve of Lp(a) concentration in mg/dLversus absorbance at 490 nm using anti-kringle 4 Mab 4D2 as the captureantibody and HRPO-labeled anti-kringle 5 Mab for detection as describedin Example 6.

[0036]FIGS. 13A and 13B show correlation curves for Lp(a) assays usinganti-kringle 4 Mab 4D2 as the capture antibody and HRPO-labeledanti-kringle Mab 1-892-230 for detection (y-axis) vs. the Terumo ELISA(x-axis). FIG. 13A compares the two assays using normal subjects andFIG. 13B uses patients as described in Example 6.

[0037]FIG. 14 shows a calibration curve of Lp(a) concentration in mg/dLversus absorbance at 490 nm using anti-apo(a) Pab as the captureantibody and HRPO-labeled anti-kringle 5 Mab for detection as describedin Example 7.

[0038]FIGS. 15A and 15B show correlation curves for Lp(a) assays usingsheep polyclonal anti-anti apo(a) as the capture antibody andHRPO-labeled anti-kringle Mab 1-892-230 for detection vs. the TerumoELISA. FIG. 15A compares the two assays using normal subjects and FIG.15B uses patients as described in Example 7.

[0039]FIG. 16 shows a calibration curve of Lp(a)-cholesterolconcentration in mg/dl, versus absorbance at 490 nm using anti-kringle 5Mab as the capture antibody and HRPO-labeled digitonin for detection asdescribed in Example 8.

[0040]FIG. 17 shows a correlation curve for Lp(a)-cholesterol assaysusing anti-kringle 5 Mab 1-892-230 as the capture antibody andHRPO-labeled digitonin for detection with calculated Lp(a)-cholesterollevels obtained using the TERUMO ELISA as described in Example 8.

[0041]FIG. 18 shows the amino acid sequence (SEQ ID NO:1) of Kringle 5of apo(a).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

[0042] The term “test sample”, as used herein, includes biologicalsamples which can be tested by the methods of the present invention andinclude human and animal body fluids such as whole blood, serum, plasma,cerebrospinal fluid, urine, lymph fluids, and various externalsecretions of the respiratory, intestinal and genitorurinary tracts,tears saliva, milk, white blood cells, and the like, biological fluidssuch as cell culture supernatants, fixed tissue specimens and fixed cellspecimens. Any substance which can be adapted for testing with thereagents described herein and assay formats of the present invention arecontemplated to be within the scope of the present invention.

[0043] The term “analyte”, as used herein, is the substance to bedetected which may be present in the test sample. The analyte can be anysubstance for which there exists a naturally occurring specific bindingmember (such as, an antibody), or for which a specific binding membercan be prepared. Thus, an analyte is a substance that can bind to one ormore specific binding members. Analytes include but are not limited toantigenic substances, haptens, antibodies, and combinations thereof. Theterm “anti-analyte”, as used herein, refers to an analyte specificbinding member.

[0044] A “specific binding member” or “specific binding agent”, as usedherein, refers to one member or partner of a specific binding pair. A“specific binding pair” refers to two different molecules wherein one ofthe molecules through chemical or physical means specifically binds tothe second molecule. A typical example of specific binding members oragents which constitute a specific binding pair are an antigen and anantibody. Other specific binding pairs can include biotin and avidin,carbohydrates and lectins, cofactors and enzymes, enzyme inhibitors andenzymes, effector and receptor molecules, and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, antibodies, antibody fragments, both monoclonal andpolyclonal, and complexes thereof.

[0045] The term “ancillary specific binding member”, as used herein,refers to a specific binding member which binds to an analyte specificbinding member and includes for example, an antibody to an antibody.

[0046] The term “hapten” as used herein, refers to a partial antigen ornon-protein binding member which is capable of binding to an antibody,but which is not capable of eliciting antibody formation unless coupledto a carrier protein.

[0047] A “capture reagent” as used herein, refers to an unlabeledspecific binding member which is specific either for the analyte as in asandwich assay, for the indicator reagent or analyte as in a competitiveassay, or for an ancillary specific binding member, as in all indirectassay. The capture reagent can be directly or indirectly bound to asolid phase material before the performance of the assay or during theperformance of the assay, thereby enabling the separation of immobilizedcomplexes from the test sample.

[0048] An “indicator reagent” as used herein comprises a specificbinding member conjugated to a label. Indicator reagents include labeledspecific binding members which directly bind to analytes of interest andlabeled ancillary specific binding members.

[0049] “Solid phases” (“solid supports”) are known to those in the artand include the walls of wells of a reaction tray, test tubes,polystyrene beads, magnetic beads, nitrocellulose strips, membranes,microparticles such as latex particles, sheep (or other animal) redblood cells, and Duracytes® (red blood cells “fixed” by pyruvic aldehydeand formaldehyde, available from Abbott Laboratories, Abbott Park, Ill.)and others. The “solid phase” is not critical and can be selected by oneskilled in the art. Thus, latex particles, microparticles, magnetic ornon-magnetic beads, membranes, plastic tubes, walls of microtiter wells,glass or silicon chips, sheep (or other suitable animal's) red bloodcells and Duracytes® are all suitable examples. Suitable methods forimmobilizing peptides on solid phases include ionic, hydrophobic,covalent interactions and the like. A “solid phase”, as used herein,refers to any material which is insoluble, or can be made insoluble by asubsequent reaction. The solid phase can be chosen for its intrinsicability to attract and immobilize the capture reagent. Alternatively,the solid phase can retain an additional receptor which has the abilityto attract and immobilize the capture reagent. The additional receptorcan include a charged substance that is oppositely charged with respectto the capture reagent itself or to a charged substance conjugated tothe capture reagent. As yet another alternative, the receptor moleculecan be any specific binding member which is immobilized upon (attachedto) the solid phase and which has the ability to immobilize the capturereagent through a specific binding reaction. The receptor moleculeenables the indirect binding of the capture reagent to a solid phasematerial before the performance of the assay or during the performanceof the assay. The solid phase thus can be a plastic, derivatizedplastic, magnetic or non-magnetic metal, glass or silicon surface of atest tube, microtiter well, sheet, bead, microparticle, chip, sheep (orother suitable animal's) red blood cells, Duracytes® and otherconfigurations known to those of ordinary skill in the art.

[0050] It is contemplated and within the scope of the present inventionthat the solid phase also can comprise any suitable porous material withsufficient porosity to allow access by detection antibodies and asuitable surface affinity to bind antigens. Microporous structuregenerally are preferred, but materials with gel structure in thehydrated state may be used as well. Such useful solid supports includebut are not limited to nitrocellulose and nylon. It is contemplated thatsuch porous solid supports described herein preferably are in the formof sheets of thickness from about 0.01 to 0.5 mm, preferably about 0.1mm. The pore size may vary within wide limits, and preferably is fromabout 0.025 to 15 microns, especially from about 0.15 to 15 microns. Thesurface of such supports may be activated by chemical processes whichcause covalent linkage of the antigen or antibody to the support. Theirreversible binding of the antigen or antibody is obtained, however, ingeneral, by adsorption on the porous material by poorly understoodhydrophobic forces. Other suitable solid supports are known in the art.

[0051] The term “label”, as used herein, refers to any substance whichcan be attached to specific binding agents, such as antibodies,antigens, cholesterol binding agents, Lp(a) specific binding agents andanalogs thereof, and which is capable of producing a signal that isdetectable by visual or instrumental means. Various suitable labels foruse in the present invention can include chromogens, catalysts,fluorescent compounds, chemiluminescent compounds, radioactive elements,colloidal metallic (such as gold), non-metallic (such as selenium) anddye particles (such as the particles disclosed in U.S. Pat. Nos.4,313,734, 4,954,452, and 4,373,932), enzymes, enzyme substrates, andorganic polymer latex particles (as disclosed in co-owned U.S. Pat. No.5,252,459, issued Oct. 12, 1993), liposomes or other vesicles containingsuch signal producing substances, and the like. A large number ofenzymes suitable for use as labels are disclosed in U.S. Pat. No.4,275,149. Such enzymes include phosphatases and peroxidases, such asalkaline phosphatase and horseradish peroxidase which are used inconjunction with enzyme substrates, such as nitro blue tetrazolium,3,5′,5,5′-tetranitrobenzidine, 4-methoxy-1-naphthol,4-chloro-1-naphthol, 5-bromo-4-chloro-3-indolyl phosphate,chemiluminescent enzyme substrates such as the dioxetanes described inU.S. Pat. Nos. 4,857,652 (issued Aug. 15, 1989), 4,931,223 (issued Jun.5, 1990), 4,931,569 (issued Jun. 5, 1990), 4,962,192 (issued Oct. 9,1990), and 4,978,614 (issued Dec. 18, 1990), and derivatives and analogsthereof. Fluorescent compounds such as fluorescein, phycobiliprotein,rhodamine and the like, including their derivatives and analogs aresuitable for use as labels.

[0052] The linking of labels, i.e. labeling of peptides and proteins iswell known to those of ordinary skill in the art. For example,monoclonal antibodies produced by a hybridoma can be labeled bymetabolic incorporation of radioisotope-containing amino acids providedas a component in the culture medium. (See, for example, Galfre et al.,(1981) Meth. Enzymol., 73: 3-46). The techniques of protein conjugationor coupling through activated functional groups are particularlyapplicable. (See, Avrameas et al., (1978) Scand. J. Immunol., 8(7):7-23. Rodwell et al. (1984) Biotech., 3: 889-894 and U.S. Pat. No.4,493,795).

[0053] The term antibody is also meant to include both intact moleculesas well as fragments thereof, such as, for example, Fab and F(ab′)₂which are capable of binding antigen. Fab and F(ab′)₂ fragments lack theFc fragment of intact antibody and may have less non-specific bindingthan an intact antibody (Wahl, et al., J. Nucl. Med. 24: 316-325, 1983),as well as increased kinetics due to their smaller size. Such fragmentsalso may be used for the detection and quantitation of lipoproteincholesterol particles according to the methods disclosed herein in thesame manner as intact antibodies. Such fragments are well known in theart and are typically produced by enzymatic degradation of an antibody,such as with pepsin, papain, or trypsin. Alternatively, antibodies andantibody fragments can be prepared using recombinant antibody methodssuch as those described in U.S. patent applications Ser. Nos. 513957,693249, 789619, 776391, 799770, 799772, and 809083, wherein antibodiesor antibody fragments are produced from the RNA of an antibody producingB-cell from an immunized animal, such as a rat, mouse, rabbit or human,using known recombinant techniques.

[0054] Kringle 5 specific binding agents according to the presentinvention also include bacteriophage described in U.S. Pat. No.4,797,363. Bacteriophage tail or head segments are capable ofselectively binding antigens. By mutation and selection processes,bacteriophage having the necessary binding characteristics toselectively bind lipoprotein cholesterol particles can be obtained.

[0055] Kringle 5 specific binding agents according to the presentinvention also include nucleic acid sequences, such as DNA and RNA,which selectively bind to Lp(a) particles. A library of nucleic acidsequences are tested for the desired binding characteristics and thesequences that are specific for lipoprotein (a) particles are isolatedand replicated. Weintraub, et al., WO 92/05285, and Gold, et al., W091/19813, disclose methods for the preparation of DNA and RNA sequencewhich are antigen specific.

The Invention

[0056] The present invention provides a method for the detection andquantitation of Lp(a) in a fluid sample. A binding agent specific for akringle 5 epitope(s) of apo(a) is used to capture intact Lp(a) particlesfrom a fluid sample, preferably a plasma sample. The amount of Lp(a)present in the plasma sample is then determined from the amount of Lp(a)in the binding agent-Lp(a) complexes formed in the reaction. The presentinvention also provides reagents, such as kringle 5 specific bindingagents which preferably are monoclonal antibodies, for use in themethods described herein.

[0057] The claimed method utilizes a kringle 5 specific binding agent toform a binding complex with Lp(a) particles in a sample. In oneembodiment, the method is performed by combining all components of thetest mixture simultaneously i.e. a binding agent specific for kringle 5of apo(a), a test sample, and any indicator reagent(s) for detectingLp(a)) and then determining the amount of Lp(a) present in the bindingagent-Lp(a) complexes. In a second embodiment, a test sample is combinedwith a kringle 5 specific binding agent and then separated from thebinding agent-Lp(a) complexes formed before measuring the amount ofLp(a) in the complex. Preferably, the Lp(a) particles are captured by akringle 5 specific binding agent directly or indirectly bound to a solidsupport. This methodology simplifies the separation of the resultingbinding agent-Lp(a) complexes.

[0058] Kringle 5 specific binding agents include kringle 5 specificbinding proteins, such as monolonal (Mab) and polyclonal antibodies(Pab) and other kringle 5 specific synthetic or recombinant proteinsthat specifically bind to kringle 5 of apo(a) or a port thereof (i.e. adomain). A binding agent that specifically binds kringle 5 of apo(a)will bind to substantially all Lp(a) via a kringle 5 domain of apo(a) inLp(a) particles and will not cross-react, e.g. exhibit less than about2% cross-reactivity with plasminogen and with other lipoproteinparticles, such as LDL, very low density lipoprotein (VLDL),intermediate density lipoprotein (IDL), and high density lipoprotein(HDL). In more preferred embodiment, a kringle 5 specific binding agentis one which binds to substantially all Lp(a) via kringle 5 of apo(a),to plasminogen at less than 1% of Lp(a) binding and to LDL, VLDL, IDLand HDL at less than 2% of Lp(a) binding. In a most preferredembodiment, a kringle 5 specific binding agent exhibits no detectablecross-reactivity to other lipoproteins or to plasminogen. An antibody isa preferred kringle 5 specific binding agent and a monoclonal antibodythe most preferred.

[0059] A kringle 5 specific binding agent is preferably attacheddirectly or indirectly to a solid support, for example, by absorption,adsorption, covalent coupling directly to the support or indirectlythrough another binding agent (such as a second antibody), or the likeutilizing methods known in the art. The type of attachment or bindingwill typically be dependent upon the material composition of the solidsupport and the type of Lp(a) specific binding agent used in the assay.For example, nitrocellulose, polystyrene and similar materials possesschemical properties that permit absorption or adsorption of proteins toa solid phase composed of this material. Other materials, such as latex,nylon, and the like contain groups that permit covalent coupling of thelipoprotein specific binding agent to the solid support. Chemical groupssuch as amines and carboxylic acids are coupled through the activationof the carboxylic acid group with, for examples carbodiimide compounds,to form an amide linkage. Other linking methods are well-known in theart particularly for coupling proteins to solid phases and oneskilled-in-the-art can easily conceive of a variety of methods forcovalently coupling the specific binding agent to the solid support. Thesolid support can take the form of a variety of materials, for example,the solid support may be in the form of a bead particle, a magneticparticle, a strip or a layered device.

[0060] Preferably, the specific Lp(a) particles of interest areseparated from other lipoprotein particles in the sample before thedetermination of the amount of Lp(a) bound to the kringle 5 specificbinding agent. The separation of the binding agent-Lp(a) complexes fromthe sample or more specifically from the other lipoprotein particles inthe sample can be accomplished in a variety of ways. When the bindingagent is coupled to a solid support, the solid support can be removedfrom the sample or the sample can be removed from the solid support. Forexample, when the solid support is a microtiter plate or another type ofreaction well device, such as the devices described in U.S. Pat. Nos.5,075,077 and 4,883,763, issued Dec. 24, 1991 and Nov. 28, 1989respectively, and U.S. patent application Ser. No. 523,629, the samplecan be removed from the wells and the plate washed of any residualsample. When the solid support is a particle, such as a latex ormagnetic particle, the solid support can be separated from the sample byfiltration through a fiber matrix, such as the devices described in U.S.Pat. No. 4,552,839, issued Nov. 12, 1985, U.S. Pat. No. 5,006,309,issued Apr. 9, 1991, EP Application 0288793, published Nov. 2, 1988, PCTPublication No. WO92/08738, published May 29, 1992. EP Patent 0424633,published Jan. 17, 1996 and Fiore et al. (1988) Clin. Chem. 34(9):1726-1732, or by attraction to a magnet followed by removal of theparticles or the sample. Alternatively, the binding agent-Lp(a)complexes can be separated or removed by filtration such as by the IonCapture Methodology described in EP Patents 0326100 and 0406473,published Sep. 11, 1996 and Sep. 20, 1995, respectively. Theseapplications describe the use of ion capture separation, in whichspecific binding members used in an assay are chemically attached to afirst charged substance and a porous matrix having bound thereto asecond charged substance that binds to the first charged substance. Aspecific binding pair is formed and separated from the reaction mixtureby an electrostatic interaction between the first and second chargedsubstances. The specific binding member is preferably covalently coupledto the first charged substance. Examples of charged substances includeanionic and cationic monomers or polymers, such as polymeric acids, e.g.polyglutamic acid, polyaspartic acid, polyacrylic acid and polyaminoacids; proteins and derivative proteins, such as albumin; anionicsaccharides, such as heparin or alginic acid; polycations, such asGafQuat™ L-200 and Celquat™ II-100. The art is replete with examples ofsolid supports, as well as techniques in the separation of samples fromsolid supports.

[0061] Alternatively, the methods of the present invention may beperformed without the need for a separation step, as described in PCTPublication No. WO94/20636, published Sep. 15, 1994. PCT Publication No.WO94/20636 teaches genetically engineered proteins, such as hybridenzymes and their preparation and use in quantitative and qualitativeassays. In the method systems described, a hybrid enzyme is providedwhich comprises a starting enzyme and a foreign amino acid moiety thateither replaces or is inserted into an amino acid sequence of thestarting enzyme at a region close to the enzyme's active site. Theforeign moiety may be either a first member of a specific binding pairor a linking moiety to which a ligand may be coupled or conjugated. Ineither case, the resulting hybrid enzyme exhibits the enzymatic activityof the starting enzyme. Furthermore, the foreign moiety of the hybridenzyme can still bind to its corresponding specific binding pair memberor to an anti-ligand and as a consequence of such binding, modulate ormodify the activity of the hybrid enzyme. Thus, in an assay systemcomprising a hybrid enzyme, the enzymatic activity will change dependingupon the presence or the amount of analyte in the test sample.

[0062] The hybrid enzyme provides a basis for assays to detect, (1) thepresence or the amount of an antibody directly or (2) the presence orthe amount of an antigen indirectly by competition for binding to abinding molecule. One assay system which utilizes a hybrid enzymecomprises the steps of (1) contacting a test sample containing ananalyte of interest, a hybrid enzyme capable of binding to the analyteand a binding molecule of the analyte to form a reaction mixture; (2)contacting the reaction mixture with a substrate for the startingenzyme; and (3) monitoring the change, if any, in enzymatic activity ofthe hybrid enzyme. As an example, in the case of an Lp(a) competitiveassay, the monoclonal antibodies of the present invention may be used asa binding molecule of the analyte. Other assay formats, such as a directassays are also envisioned. As indicated above, the manner of makinghybrid enzymes and using them in competitive and direct immunoassays isfully described in WO94/20636.

[0063] The amount of Lp(a) in a plasma sample can be determined by avariety of assay formats. A preferred assay format, for example, is asandwich assay. This method comprises contacting a test sample with asolid phase (hereinafter represented by the symbol “|-”) to which atleast one capture reagent (i.e. anti-analyte) is bound, to form amixture. The mixture of test sample and capture reagent bound to a solidphase is incubated for a time and under conditions sufficient to allow|-capture reagent/analyte complexes to form. These complexes then arecontacted with an indicator reagent comprising a second anti-analytepreviously conjugated to a label. This second mixture is incubated for atime and under conditions sufficient for |-capturereagent/analyte/indicator reagent complexes to form. The presence of the|-capture reagent/analyte/indicator reagent complexes is determined bydetecting the measurable signal generated. In such an assay, the capturereagent bound to the solid support may be, for example, a first antibodywhich binds to an antigen in the test sample, and the indicator reagentmay be a second antibody which also binds to the antigen but at a sitedifferent from the first antibody. It is also within the scope of thepresent invention to use one antibody as a capture agent and a fragmentof an antibody as an indicator reagent. In addition, sandwich-typeassays may be configured in a reverse orientation to that describedabove, i.e. with an antigen serving as the capture reagent to test forthe presence of antibody in a test sample. In this case, the indicatorreagent is a second labeled antibody or fragment thereof which alsobinds to the complex of antigen/antibody bound to a solid support.

[0064] Detection of complexes formed in sandwich and other assays may beperformed indirectly. In an indirect sandwich assay format, complexes of|-capture reagent/analyte/second capture reagent are formed, none ofwhich are labeled. Instead, an ancillary specific binding member whichbinds to the second capture reagent acts as the indicator reagent. Forexample, when the second capture reagent is a mouse antibody to theanalyte of interest, the complex of capture reagent/analyte/mouseantibody may be detected using an ancillary antibody which is labeled,such as labeled goat anti-mouse antibody. Furthermore, the use of biotinand antibiotin, biotin and avidin, biotin and streptavidin, and thelike, may be used to enhance the generated signal in the assay systemsdescribed herein.

[0065] For purposes of illustration, the following sandwich formats maybe utilized: in a first format, Lp(a) particles present in a plasmasample are specifically captured by a kringle 5 specific monoclonalantibody immobilized on a solid support. After removing the otherlipoprotein particles, the Lp(a) bound to the solid support isquantitated using a labeled anti-kringle 4 monoclonal antibody as anindicator reagent. A second format uses similar capture phase technologyas in format 1 above, but the detection antibody in the sandwich isinstead an anti-apo(a) polyclonal antibody that is directed towards bothkringle 4 and kringle 5 domains of apo(a). In a third format, Lp(a)particles present in a plasma are captured by a kringle 4 specificmonoclonal antibody immobilized on a solid support. After washing awaythe other lipoprotein particles, the Lp(a) bound to the solid support isquantitated using a labeled anti-kringle 5 specific monoclonal antibody.In a fourth format, an anti-apo(a) kringle 4 monoclonal antibody boundto a support is used to capture Lp(a) particles and the detectionantibody is a polyclonal labeled antibody, directed towards both kringle4 and kringle 5 domains. In yet another format, Lp(a) particles arespecifically captured by a kringle 5 specific monoclonal antibody andthe bound Lp(a) is detected by another kringle 5 monoclonal antibodywith different epitope specificity, as described in the presentinvention. Preferably, in these formats, the indicator reagent islabeled with an enzyme.

[0066] Alternatively, a kringle 5 specific binding agent can be used ina sandwich immunoassay method for the quantitation of Lp(a)-cholesterolin a plasma sample. This involves the specific capture of the Lp(a)particles in the plasma sample by the kringle 5-specific antibodyimmobilized on the solid support followed by quantitation of cholesterolin the captured Lp(a) particles by a cholesterol binding agent which iscoupled directly or indirectly to a label. The Lp(a)-cholesterol boundcholesterol binding agent is then quantitated by detection andmeasurement of the label. Methods for determining cholesterol associatedwith lipoproteins are well known to those of ordinary skill in the art.(See for example, PCT Publication No. WO93/11067, published Sep. 16,1993).

[0067] In addition to the foregoing sandwich assay formats, competitiveassays are also contemplated by the invention. In one format, labeledLp(a) may compete with the Lp(a) to be determined in the plasma samplefor binding to a kringle 5 specific monoclonal antibody which has beenimmobilized on a solid support. In a second format, Lp(a) may beattached to a solid support, and then incubated with a fixed amount ofkringle 5 monoclonal antibody added to a sample suspected of containingLp(a). The amount of kringle 5 which binds to the Lp(a) on the solidsupport may then be determined using a labeled antibody which binds tothe kringle 5 antibody, for example, an anti-mouse labeled antibody. Ina third format, the Lp(a) in the sample competes with Lp(a) attached tothe solid support for binding by a labeled kringle 5 antibody. It isfully expected that other known assay formats may be advantageouslyadopted by the skilled artisan and these are within the scope of theinvention, to be utilized with the unique antibodies herein set forthand described.

[0068] Another alternative is based on an immunochromatographic assayformat (such as described in U.S. Pat. No. 4,954,452 and U.S. Pat. No.5,229,073, for example) in which the lipoprotein particles in the testsample bind to a labeled Lp(a) binding agent. The resulting complexesthen travel along a test strip by capillary action. The labeled Lp(a)complexes are then captured by a high affinity anti-Lp(a) specificantibody immobilized on the test strip, followed by detection andmeasurement of the captured labeled Lp(a) complexes. Typically, the teststrip is comprised of a porous or bibulous membrane and the result isdetermined by a visual readout of a detectable signal. Other test stripassay formats are also within the scope of the invention.

[0069] The use of scanning probe microscopy (SPM) for immunoassays alsois a technology to which the monoclonal antibodies of the presentinvention are easily adaptable. In scanning probe microscopy, inparticular in atomic force microscopy, in the capture phase, forexample, at least one of the monoclonal antibodies of the invention isadhered to a solid phase and a scanning probe microscope is utilized todetect antigen/antibody complexes which may be present on the surface ofthe solid phase. The use of scanning tunneling microscopy eliminates theneed for labels which normally must be utilized in many immunoassaysystems to detect antigen/antibody complexes. Such a system is describedin Publication No. WO 92/15709, published Sep. 17, 1992.

[0070] The use of SPM to monitor specific binding reactions can occur inmany ways. In one embodiment, one member of a specific binding pair(described below) is attached to a surface suitable for scanning. Theattachment of the specific binding member may be by adsorption to a testpiece comprising a solid phase of a plastic or metal surface, usingmethods known to those of ordinary skill in the art.

[0071] Alternatively, a specific binding member may be covalently (i.e.irreversably) attached to a test piece, in which case the test piececomprises a solid phase of derivatized plastic, metal, silicon, orglass. Covalent attachment methods are also known to those skilled inthe art. If the test piece is silicon or glass, the surface must beactivated prior to attaching the specific binding member. Activatedsilane compounds such as triethoxy amino propyl silane (available fromSigma Chemical Co., St. Louis, Mo.), triethoxy vinyl silane (AldrichChemical Col. Milwaukee, Wis.) and (3-mercapto-propyl)-trimethoxy silane(Sigma Chemical Co., St. Louis, Mo.) can be used to introduce reactivegroups such as amino-, vinyl-, and thiol-, respectively. Such activatedsurfaces can be used to link the binding member directly (in the casesof amino or thiol) or the activated surface can be further reacted withlinkers such as glutaraldelhyde, bis (succinimidyl), SPPD 9 succinimidyl3-[2-pyridylthio]propionate), SMCC(succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate). SIAB(succinimidyl-[4-iodoacetyl]aminobenzoate, and SMPB (succinimidyl4-[1-maleimidophenyl]butyrate) to separate the binding member from thesurface. The vinyl group can be oxidized to provide a means for covalentattachment. It also can be used as an anchor for the polymerization ofvarious polymers such as polyacrylic acid, which can provide multipleattachment points for specific binding members. The amino surface can bereacted with oxidized dextrans of various molecular weights to providehydrophilic linkers of different size and capacity. Examples ofoxidizable dextrans include Dextran T-40 (molecular weight 40,000daltons), Dextran T-110 (molecular weight 110,000 daltons), DextrinT-500 (molecular weight 500,000 daltons), Dextran T-2M (molecular weight2,000,000 daltons), all of which are available from Pharmacia,Piscataway, N.J., or Ficoll (molecular weight 70,000 daltons), availablefrom Sigma Chemical Co. Also, polyelectrolyte interactions may be usedto immobilize a specific binding member on a surface of a test peice byusing techniques and chemistries described supra in the Ion Capturemethodology. Attachment by covalent means is a preferred method.

[0072] Following attachment of a specific binding member, the surfacemay be further treated with materials such as serum, proteins, or otherblocking agents to minimize non-specific binding. The surface also maybe scanned either at the site of manufacture or point of use to verifyits suitability for assay purposes. The scanning process is notanticipated to alter the specific binding properties of the test piece.

[0073] Also included as part of the invention are immunoassay kits forthe detection and quantification of Lp(a) in a patient sample whichincludes one or more of the heretofore described labeled reagents andcapture reagents. For example, it is contemplated that the reagentemployed for the assay can be provided in the form of a kit with one ormore containers such as vials or bottles, with each container containinga separate reagent such as a monoclonal antibody, or a cocktail ofmonoclonal antibodies employed in the assay. These kits also couldcontain vials or containers of other reagents needed for performing theassay, such as washing, processing and indicator reagents.

[0074] The following examples are illustrative of the invention and arein no way to be interpreted as limiting the scope of the invention asdefined in the claims. It will be appreciated that oneskilled-in-the-art can conceive of many other devices and methods foruse of which the present inventive concepts can be applied. Throughoutthe entire specification, it is intended that citations to theliterature (whether patents, patent applications or articles) areexpressly incorporated by reference.

GENERAL METHODOLOGIES

[0075] 1. Development of Anti-Kringle Monoclonal Antibodies:

[0076] Peptide sequences of the kringle 4 repeats and the single kringle5 domain in apo(a) are well known (Eaton et al. (1987) Proc Natl AcadSci 84: 3224-3228; McLean et al. (1987) Nature (London) 330: 132-137).Recently, Li et al. have expressed and purified kringle 4 in Escherichiacoli (Li et al. (1992) Protein Express Purification 3: 212-222). Arecent report describes the expression of a recombinant kringle 5 ofhuman apo(a) (Chevinesse et al (1996) Protein Expression andPurification 8: 145-150). Kringle 5 of apo(a) containing 101 peptideresidues [SEQ ID NO:1 and also shown as residues 4204-4294 of FIG. 1 ofMcLean et al. (1987) supra] was expressed as a fusion protein withmaltose binding protein in Escherichia coli. The fusion protein wasfirst purified from whole cell lysate by amylose agarose affinitychromatography, then cleaved to release kringle 5 protein which waspurified by fast flow liquid chromatography (Pharmacia). The purifiedsamples of kringle 5 and kringle 5-fusion protein were used formonoclonal antibody production.

[0077] a. Immunization: Six female 4-6 week old BALC/c mice (CharlesRiver, Wilmington Mass.) were immunized with the kringle 5 fusionprotein of lipoprotein (a) (Lp(a)) at week 0, 4, 8, 16, and 20. The doselevel was 12.5 mg in 0.1 mL using 1:1 ratio of the kringle 5 fusionprotein with RIBI adjuvant (RIBI Immunochem Research, Inc, Hamilton,Mont.). The adjuvant emulsion route of injection was equally distributedinterperitoneally and subcutaneously. Three days prior to the fusion,mice were given an immunization of 12.5 mg Kringle 5 fusion protein viaintersplenic injection.

[0078] b. Sera Evaluation: Ten days following the fifth immunization,sera samples were taken by retro-orbital vein puncture. Sera sampleswere analyzed for Lp(a) specific antibody titer by enzyme immunoassay(EIA). Microtiter wells were coated with 100 mL of Lp(a) at 1 mg/mL inphosphate buffered saline (PBS) or 100 mL PBS and incubated at roomtemperature overnight. The following day the plates were blocked for 30minutes with 200 mL per well of 3% v/v fish gelatin (Norland ProductsInc., New Brunswick, N.J.) in PBS. After washing the plate, 50 mL ofserum was added per well, at log 2 serial dilutions starting at a 1:100dilution, and incubated 1 hour. The plates were washed and 50 mL ofdiluted goat anti-mouse IgG+M-HRPO (Kirkegaard and Perry Laboratories,Gaithersburg, Md.), was added per well to the plate for a 30 minuteincubation period. The plate was washed a final time and the substrateo-phenylenediame 2HCI (OPD) (Abbott Laboratories, Abbott Park, Ill.) wasadded to develop the color. The relative intensity of optical densityreadings identified mouse number 2 & 6 to have the highest Lp(a) titerwith minimal non-specific reactivity and these mice were selected forfusion twenty nine weeks following the first immunization.

[0079] c. Fusion: On the day of fusion, the two mice were euthanized bycervical dislocation and a splenectomy was performed. Splenocytes wereflushed out and washed in Iscoves's Modified Dulbecco's Medium (IMDM)(GIBCO, Grand Island, N.Y.) and centrifuged at 1000 rpm for 5 minutes.The splenocytes were combined with SP2/0 myeloma cells at a 2:1 ratio,washed in IMDM, and centrifuged. The supernatant was removed and 1 mL of50% polyethylene glycol (PEG) (American Type Culture Collection,Rockville, Md.) was added to the pellet for one minute as the pellet wasgently dispersed by tapping and swirling. Thirty mLs of IMDM were addedto the mixture and centrifuged as previously described. The supernatewas decanted and the pellet resuspended in IMDM with HAT (HypoxanthineAminopterin Thymidine) (GIBCO, Gaithersburg, Md.), 15% Fetal BovineSerum (FBS) (Hyclone Laboratories, Logan, Utah), Origen HybridomeCloning Factor (Igen, Rockville, Md.), and Salmonella typhimuriummitogen (STM) (1% v/v) (RIBI Immunochem Research, Inc. Hamilton, Mont.).The fused cells were plated into 96 well tissue culture plates at 3×10⁵cells per well. The cell culture media was changed by asepticallyaspirating half the tissue culture supernate and feeding with IMDM with1% v/v HT (hypoxanthine and thymidine) Supplement (GIBCO, Gaithersburg,Md.), and 10% v/v FBS at days five and seven. The fusion protocol wasreferenced from Galfre, G. and Milstein, C. (1981). Preparation ofMonoclonal Antibodies: Strategies and Procedures, Meth Enzymol 73: 1-46.

[0080] d. Fusion Screening: The primary screening of the fusion occurredon day ten with confluent cultures. An EIA was run similar to the assayused to test sera samples. Microtiter wells were coated with 100 mL of 1mg/mL Lp(a) in PBS and incubated at room temperature overnight. Afterwashing and blocking, as previously described, 50 mL of culturesupernate was added and incubated 1 hour. The plates were washed andgoat anti-mouse HRPO conjugate was added to each well. This was followedby washing and color development with OPD. The relative intensity ofoptical density readings identified hybrids 1-292, 1-390, 1-431, 1-458,1-532, 1-546, 1-746, & 1-892 as 3 times that of negative control, normalmouse serum (NMS) (Organon Teknika-Cappel, Malvern, Pa.). These hybridswere then expanded. All the above listed hybrids were retested using thesame EIA format described previously. These hybrids were selected forcloning because the optical density readings indicated specific bindingto Lp(a) with minimal nonspecific binding.

[0081] c. Hybrid Cloning: Hybrids 1-292, 1-390, 1-431, 1-458, 1-532,1-546, 1-746 and 1-892 were cloned by limiting dilutions starting at1:100, 10-fold to 10⁶. The cloning media used was IMDM with 10% v/v FBSand 1% v/v HT Supplement. A 200 mL cell suspension was added to each ofthe 96 wells in the TC plate.

[0082] f. Clone Selection: The clone screening occurred on day ten withconfluent cultures. Clones 1-292-189, 1-390-191, 1-431-378, 1-458-165,1-532-266, 1-546-264, 1-746-183, and 1-892-230 were selected based onEIA reactivity specific to Lp(a) with minimal non-specific binding. TheEIA screening protocol used was as described previously.

[0083] g. Isotypes: The isotypes of the monoclonal antibodies secretedfrom the cell lines identified above were determined using an EIAclonotyping kit (Southern Biotech, Birmingham, Ala.). The assay wasperformed according to the manufacturer's recommendations and theresults are shown below. Monoclonal Type Monoclonal Type 1-292-189 IgMkappa 1-390-191 IgG3 kappa 1-431-378 IgM kappa 1-458-165 IgG2a kappa1-546-264 IgM kappa 1-532-266 IgG2a kappa 1-746-183 IgM1 kappa 1-892-230IgG1a kappa

[0084] h. Antibody Production: Cell lines with an IgG isotype wereexpanded in tissue culture flasks using IMDM with 5% v/v Fetal CalfSerum (Hyclone Laboratories, Logan, Utah) at a cell density between1×10⁴ cells/mL and 5×10⁵ cells/mL until they could be expanded intoroller bottles. The cells were allowed to grow in the roller bottles formaximum antibody production, normally until viability fell below 5%.

[0085] Cell lines with an IgM isotype were expanded in tissue cultureflasks with IMDM with 5% v/v Fetal Calf Serum at a cell density between1×10⁴ cells/mL and 5×10⁵ cells/ml, with viability >90%. These cells wereused for ascites production in BALB/c mice as described by Brodeur etal., Production of monoclonal antibodies in mouse ascites, in MonoclonalAntibody Production Techniques and Applications (L. Schook, ed.), MarcelDekker, Inc., New York, 1987, pp. 99-111.

[0086] i. Antibody Purification: Cultures were removed from rollerbottles and the cells were allowed to settle for three days at 4-8° C.Cell supernate was filtered through a 0.45 mm filter and concentratedapproximately 20-fold using an Amicon Concentrator, (Amicon Corp.,Beverly, Mass.). The concentrated supernate was filtered through anadditional 0.45 mm filter. This material was then purified by Protein ASepharose column chromatography as described by Ey et al., “Isolation ofPure IgG1, IgG2a, and IgG2b immunoglobulins from Mouse Serum usingProtein A Sepharose”, Immunochem 15: 429-436 (1978). The purified anddialyzed antibody was tested for Lp(a) reactivity by EIA as previouslydescribed.

[0087] Ascites fluid was filtered through a 0.22 mm filter and purifiedby gel filtration on a Sephacryl S-300 (Pharmacia LKB, Piscataway, N.J.)sizing column as described by Bouvet et al., “A Modified Gel FiltrationTechnique Producing Unusual Exclusion Volume IgM: a Simple Way ofPreparing Monoclonal IgM” Journal of Immunological Methods 66: 299-305(1984). The purified antibody was tested for Lp(a) reactivity by ELISAas previously described.

[0088] 2. Evaluation of the Monoclonal Antibodies

[0089] a. Direct ELISA using lipoprotein and kringle 5 coated microtiterplates: Lipoprotein fractions (LDL, HDL, VLDL, IDL, and Lp(a)), purifiedby ultracentrifugation (see Example 2, infra), and purified kringle 5,obtained from Dr. Gunther Fless of the University of Chicago, werecoated on separate wells of a Maxisorb Nunc Immuno Plate as follows: onehundred microliters (100 mL) of each lipoprotein fraction at alipoprotein-cholesterol concentration of about 1 mg/mL and 1 mg/mL ofkringle 5 in 20 mM phosphate buffered saline, pH 7.0 (PBS) weredispensed into separated wells of the microtiter plate. The plate wasincubated at 37° C. for one hour, then washed five times with PBScontaining 0.05% (v/v) Tween 20 (PBS-Tween 20). The non-specific bindingsites were blocked by incubating 200 mL of 10% (v/v) fetal bovine serum(FBS) in PBS in each well at 37° C. for one hour and then the wells werewashed five times with PBS-Tween 20. Each Mab was diluted in 3% (v/v)FBS in PBS to a final antibody concentration of about 2 mg/mL and thediluted Mab solutions were then serially diluted in the reaction wellsof the plate. After incubation at 37° C. for one-half hour, the platewas washed five times with PBS-Tween 20. Thereafter one hundredmicroliters (100 mL) of horseradish peroxidase (HRPO) labeled goatanti-mouse IgG+IgM (obtained from Kirkegaard and Perry Laboratories,MD), diluted in 3% FBS in PBS to a final concentration of about 1.25mg/mL, were added to each reaction well and the plate was incubated at37° C. for one-half hour. The plate was then washed eight times withPBS-Tween 20. One hundred microliters (100 mL) of freshly prepared HRPOsubstrate solution, containing one o-phenylenediamine (OPD) tablet perfive milliliters (5 mL) of citrate buffer, pH 6 (both available fromAbbott laboratories, IL), were added to each well. The color reactionwas stopped after five minutes by adding 100 mL of 1N H₂S0₄ to thereaction wells. An absorbance reading of each reaction well was thenobtained with a Bio-Tek microplate reader at 490 nm. Typical bindingcurves for each lipoprotein and kringle 5 tested with Mab 1-892-230 areshown in FIG. 1. As can be seen from these binding curves, the Mab bindsonly to Lp(a) and kringle 5 and not to any other lipoproteins. FIGS. 2Aand 2B show a comparison of the binding of four selected IgG Mabs andfoul selected IgM Mabs developed against the kringle 5 immunogen. Theresults show that three IgG Mabs 1-892-230, 1-532-266 and 1-458-165 havesimilar reactivities for Lp(a). In the IgM Mab series, Mab 1-746-183showed higher reactivity for Lp(a) when compared to 1-292-189, and muchhigher reactivity than the other two Mabs.

[0090] b. Direct ELISA using Mab-coated microtiter plates: Mabs werecoated onto the reaction wells of microtiter plates after dilution ofthe Mabs in PBS as follows: IgG Mabs 1-892-230, 1-532-266, 1-390-191 and1-458-165 were each diluted to a concentration of 20 mg/mL; IgM Mabs1-746-183, 1-546-264, 1-431-378 and 1-292-189 were each diluted to aconcentration of 50 mg/mL. One hundred microliters (100 mL) of each Mabsolution were dispensed into separate reaction wells and incubated atroom temperature on a rotator at 100 rpm for two hours. The plates werethan washed five times with PBS-Tween 20 and blocked with 200 mL of 10%FBS in PBS by incubation at 37° C. for one hour. The plates were thenwashed five times with PBS-Tween 20.

[0091] Lp(a) was then serially diluted in PBS into each Mab plate,starting with an Lp(a) protein concentration of 5 mg/mL, so that eachwell contained a total of 100 mL of solution. After incubation at 37° C.for one-half hour, the plates were washed five times with PBS-Tween 20.One hundred microliters (100 mL) of 0.5 mg/mL Mab 4D2-HRPO or 5 mg/mLsheep Pab-HRPO conjugate (prepared according to Example 3) in 3% FBS inPBS were added to each well and incubated at 37° C. for one-half hour.(The specificitites of both labeled Abs are discussed in section 2d.below.) HRPO substrate was added and the absorbance measured asdescribed in section 2a above. The results indicated that among the IgGMabs only the Mab 1-892-230 gave a positive reaction, as shown in FIG.3. None of the four IgM Mabs reacted in this assay format. It should benoted that some of these same IgG and IgM Mabs did bind to Lp(a) whenLp(a) was immobilized oil the solid phrase as described in section 2aabove and FIGS. 2A and 2B. Thus, the reactivity of some Mabs towardsLp(a) is dependent oil whether the reaction is done on a solid phase orin a fluid phase.

[0092] c. Specificity of the antibodies for Lp(a) using lipoproteincoated microliter plates in competitive assays: The specificities ofthree selected IgG Mabs, 1-892-230, 1-532-266 and 1-458-165 weredetermined by competitive binding of the Mabs to Lp(a), kringle 5 andplasminogen (American Diagnostica) in microplate wells coated withLp(a). The Lp(a)-coated plates were prepared as described previously(see Section 2a above). Each Mab was diluted with 3% (v/v) FBS in PBS toa concentration that was two times the Mab concentration at 50%Lp(a)-binding, as determined from the binding curves prepared in section2a above. Examples of such curves are shown in FIG. 2. Purified Lp(a),kringle 5 and plasminogen at starting concentrations of 1 mg/mL in PBSeach, were serially diluted with PBS in reaction wells previouslyblocked with 10% (v/v) FBS in PBS. To each of these wells were added 50mL of the diluted Mab solutions. The Mab-competitor mixtures wereincubated at room temperature for one-half hour on a rotator at 100 rpm.The contents from each well were then transferred to Lp(a)-coatedreaction wells and the plates were incubated at 37° C. for one-halfhour. The amount of Mab bound to the Lp(a)-coated reaction wells wasmeasured according to the method described in section 2a above. Thebinding curves of the three IgG Mabs are shown in FIGS. 4A, 4B and 4C.The inhibition curves for the Mab 1-892-230 in FIG. 4A indicate that thebinding of this Mab to Lp(a) can be inhibited by kringle 5 and by Lp(a)but not by plasminogen. The inhibition curves for the Mab 1-532-266 inFIG. 4B indicate that the binding of this Mab to Lp(a) can be inhibitedbetter by Lp(a) than by kringle 5 and not at all by plasminogen. On theother hand, the binding of Mab 1-458-165 to Lp(a) cannot be inhibited byLp(a) itself, but is very effectively inhibited by plasminogen andweakly inhibited by kringle 5. From these inhibition studies, it can beinferred that the above three IgG Mabs do not have the same reactivitywith and may not be directed to the same epitope(s) of kringle 5. TheMab 1-458-165 is possibly directed towards an epitope that recognizesplasminogen better than kringle 5. The Mab 1-532-266 is possiblydirected towards an epitope of kringle 5 that is more accessible onLp(a) than kringle 5. Thus, the Mab 1-892-230 appears to be an ideal Mabfor our purposes because of its inhibition by both kringle 5 and Lp(a).

[0093] d. Selection of antibodies that specifically bind to kringle 4and kringle 4/kringle 5: Monoclonal antibodies specific for the kringle4 domain were developed in our laboratory (see U.S. Pat. No. 5,229,073;Li et al. (1992) Protein Express Purification 3: 212-222). FIG. 5A showsthe binding curves of one of these Mab, 4D2. Polyclonal antibody againstapo(a), which is purified by adsorption with an LDL-Sepharose gel toremove unwanted cross-reactive antibodies, was selected for detection ofboth kringle 4 and kringle 5 domains of apo(a). The binding curve ofthis Pab is shown in FIG. 5B. To illustrate the binding of the above twoantibodies with the kringle 5 domain of apo(a), the following experimentwas performed: Kringle 5 (1 mg/mL) in PBS was coated onto the wells of amicrotiter plate and then the wells were blocked with 10% (v/v) FBS asdescribed in Section 2a above. The anti-kringle 4 Mab 4D2 and theanti-apo (a) Pab were then serially diluted starting at 100 mg/mL. Theexperiment was completed as previously described in section 2a exceptthat the wells containing Mab 4D2 received HRPO-labeled goat anti-mouseIgG (Kirkegaard and Perry Laboratories, MD) and the wells containing Pabanti-apo (a) received HRPO-labeled rabbit anti-sheep IgG, both at aconcentration of 0.5 mg/mL in 3% (v/v) FBS in PBS. The binding curves ofthe two antibodies are shown in FIG. 6. The results demonstrate that Mab4D2 does not bind to kringle 5, indicating that its previously definedspecificity against the kringle 4 domain has no cross-reactivity withthe kringle 5 domain, and that polyclonal anti-apo(a) binds to kringle 5as well as the previously shown binding to kringle 4.

[0094] e. Binding of anti-kringle antibodies to Lp(a) and kringle 5captured by anti-Lp(a) antibodies: The anti-kringle 4 Mab 4D2 and theanti-apo(a) Pab as described above were selected to confirm that the Mab1-892-230 binds to the kringle 5, and not the kringle 4, domain ofapo(a). For this purpose, the two antibodies were labeled with HRPO asdescribed in Example 2. The experiment was performed as follows: Mab1-892-230 (20 mg/mL) was coated onto the wells of microtiter plates andthen the wells were blocked as described in section 2c above. In oneplate Lp(a), and in the other plate, purified kringle 5 were seriallydiluted starting at 10 mg/mL. After incubation at 37° C. for one hour,the plates were washed five times with PBS-Tween 20. One hundredmicroliters (100 mL) of HRPO-labeled anti-kringle 4 Mab 4D2 (0.5 mg/mL)or HRPO-labeled anti-apo(a) Pab (5 mg/mL) in 3% (v/v) FBS in PBS wereadded to respective wells in each plate. The remainder of the procedurewas as described in section 2b above.

[0095] The results are shown in FIGS. 7A and 7B. FIG. 7A shows thatLp(a) containing both kringle domains binds to anti-kringle 4 Mab andanti-apo(a) Pab labeled Abs using anti-kringle 5 Mab as the capture Ab,confirming both kringle 4 and kringle 5 specificities. On the otherhand, the kringle 5 captured by Mab 1-892-230 as shown in FIG. 7B reactsonly with labeled anti-apo(a) Pab with kringle 4 and kringle 5specificities, but not with labelled anti-kringle 4 Mab 4D2. The aboveexperiment thus confirms that the Mab 1-892-230 is directed against anepitope of kringle 5 and does not recognize kringle 4. On the basis ofthis experiment as well as those described in Section 2a and 2b, Mab1-892-230 was selected as the best choice to develop an epitope specificLp(a) assay.

[0096] 3. Lp(a) Binding Agents

[0097] a. Lp(a) Standards: Lp(a) standards were prepared from purifiedLp(a) samples (described in Example 3 infra) by diluting in 3% (v/v) FBSin PBS for immunoassays or in 1% alkali-treated casein in PBS forsandwich assays. The Lp(a)-protein concentrations were determined andwere multiplied by 4.21 to get total Lp(a) as described by Fless et al.(1989) J. Lipid Res. 30: 651-662).

[0098] b. Preparation of Digitonin-Peroxidase Conjugates forLp(a)-Cholesterol: Three parts of a digitonin solution (2.5 mg/mL inwater) (Sigma Chemical Company, St. Louis, Mo.) were mixed with one partof a fresh solution of sodium meta-periodate (1.68%, w/v periodate inwater) at 4° C. for one hour and then the mixture was dialyzed against20 mM PBS, pH 8.0 overnight at 4° C. One part of a solution of 0.25 mMethylenediamine in 20 mM PBS, pH 8.0 was added to four parts of thedialyzed mixture and the mixture was incubated at 4° C. After 30minutes, and again after 60 minutes of incubation, 100 mL of a sodiumborohydride solution (4 mg/mL in 0.1 N NaOH) was added to the mixturefor each 30 mg of digitonin in the mixture. The mixture was thenincubated for two hours at 4° C. The resulting mixture containingethylenediamine derivatized digitonin was dialyzed against 0.01 Mcarbonate buffer, pH 9.5, at 4° C. overnight. The final carbonate buffersolution of ethylenediamine derivatized digitonin contained about 1.5 mgdigitonin/mL buffer.

[0099] Twenty-five milligrams of horseradish peroxidase (HRPO) (155Ku/mg, commercially available from Amano International) were dissolvedin 6.25 mL of water, and 1.25 mL of a freshly prepared solution of 0.2 Msodium meta-periodate was added. After 20 minutes in the dark at roomtemperature, the mixture was dialyzed against 4 liters of 1 mM acetatebuffer, pH 4.5, at 4° C. for 4 hours. The oxidized HRPO solution and theethylenediamine derivatized digitonin solultion were mixed and stirredin the dark at room temperature for two hours. Then 400 mL of a sodiumborohydride solution (4 mg/mL in water) was added and the reaction wasincubated at 4° C. After two hours, the mixture was dialyzed against 20mM PBS, pH 7.4 at 4° C. overnight. To the dialyzed reaction mixture,fatty acid free bovine serum albumin (BSA) (Sigma Chemical Company) wasadded to a final concentration of 5 mg/mL. The solution ofHRPO-digitonin conjugate was sterile filtered through a 0.22 micronfilter (Coaster Labs) and stored at −20° C.

[0100] c. Preparation of Peroxidase Conjugates of Anti-Lp(a) Antibodies:Horseradish peroxidase (155 Ku/mg, Amano International) was dissolved inwater (250 mL) and oxidized with freshly prepared 0.2 M sodiumm-periodate (50 mL) at room temperature in the dark for 20 minutes. Theoxidized peroxidase was then dialyzed against 2 liters of 1 mM acetatebuffer (pH 4.5) at 4° C. for four hours. Two mg/mL each of monoclonalantibodies against kringle 4 (4D2), against kringle 5 (1-892-230) orpolyclonal antibody against apo(a) were dialyzed against 0.01 Mcarbonate buffer (pH 9.5) at 4° C. and each was titrated with 20 mL of0.2 M carbonate buffer (pH 9.5). The antibody and the dialyzedperoxidase were then mixed at room temperature in the dark for twohours. To this mixture 24 mL of freshly prepared sodium borohydride(Aldrich, 4 mg/mL in water) was added and then incubated at 4° C. in thedark for two hours. The peroxidase-antibody conjugate was then dialyzedagainst two liters of 20 mM PBS, pH 7.4 at 4° C. and stored at −20° C.in small aliquots.

EXAMPLE 1 Preparation of Lipoprotein Fractions (LDL, VLDL, IDL and HDL)

[0101] Blood samples from normal fasting subjects were collected intoethylenediamine-tetraacetic acid (EDTA) and the red blood cells wereremoved by centrifugation. The plasma samples were then analyzed forLp(a) using the TERUMO ELISA kit (TERUMO Medical Corp., Elkton, Md.).Plasma samples containing less than 1 mg/dL Lp(a)cholesterol wereselected to use for the purification of VLDL, IDL, LDL, and HDL.Lipoprotein subfractions were prepared in a Beckman Ultracentrifuge witha SW 40 Ti rotor by successive ultracentrifugation at 4° C. (Havel etal. (1955) J. Clln. Invest. 34: 1345-1355). VLDL was collected at adensity of about 1.0006 g/mL; IDL was collected at a density range ofabout 1.006-1.019 g/mL; LDL was collected at a density range of about1.019-1.050 g/mL; and HDL was collected at a density range of about1.080-1.255 g/mL. All fractions were isolated by a tube-slicingtechnique. The lipoprotein fractions were dialyzed exhaustively against0.15 M sodium chloride containing 0.1% EDTA and 0.1% sodium azide, pH7.4 at 4° C. IDL, LDL and HDL fractions were sterile filtered through0.2 micron and VLDL through 0.45 micron membrane filters (Nalgene) andstored at 4° C.

EXAMPLE 2 Plasma Samples

[0102] The lipid profiles of 116 plasma samples from patients with noknown cardiac problems (#1-39), cardiac patients who are takinglipid-lowering drugs (#40-78) and diabetic patients (#79-116) are shownin Table 1. Total cholesterol, HDL-cholesterol and triglycerides weremeasured using an Abbott Vision® instrument and reagents (AbbottLaboratories, Abbott Park., Ill.). LDL-cholesterol (Friedewald) wascalculated using the standard equation used in the art as:[LDL-cholesterol]=[Total-cholesterol]−[HDL-cholesterol]−Triglyceride/5].LDL-cholesterol was also determined by ultracentrifugation-precipitation(Beta-quantitation) by ultracentrifugation of a plasma sample at 40,000rpm for 20 hours at a density of 1.006 gm/mL, removing the upper VLDLlayer, measuring the cholesterol and HDL-cholesterol concentrationsusing the Abbott Vision® instrument and reagents, and calculating asfollows: [LDL-cholesterol]=[infranet-cholesterol]−[HDL-cholesterol]. TheLp(a) concentrations were determined using a commercial ELISA for Lp(a)(TERUMO Medical Corp., Elkton, Md.).

EXAMPLE 3 Preparation of Lp(a) Standards and Calibrators

[0103] Lp(a) concentrations in fresh plasma samples were measured usinga commercial ELISA for Lp(a) (TERUMO Medical Corp., Elkton, Md.). Plasmasamples with high Lp(a) concentrations were ultracentrifuged for 20hours at 40,000 rpm at a density of 1.080 g/mL. The upper lipoproteinfraction containing Lp(a), LDL, VLDL and IDL was dialyzed and then theLp(a) was affinity purified on a Lp(a) specific monoclonal antibody(4F2) Sepharose 4B column as described in U.S. Pat. No. 5,229,073. Thepurity of the Lp(a) obtained from the column was determined bypolyacrylamide gel electrophoresis (PAGE) under denaturing conditions,by SDS-PAGE under reducing conditions and by Western Blot. The proteincontent of the Lp(a) obtained from the column was measured using a Lowryassay and the cholesterol concentration was measured using the AbbottVision® Cholesterol Assay (Abbott Laboratories, Abbott Park, Ill.).Lp(a) standards and calibrators were prepared from purified Lp(a) in 3%(v/v) FBS in PBS for immunoassays and in 1% alkali-treated casein in PBSfor sandwich assays.

EXAMPLE 4 Lp(a) Immunoassay with Anti-Kringle 5 Mab as Capture andHRPO-Labeled Anti-Kringle 4 Mab for Detection

[0104] a. Anti-kringle 5 Coated Plates: The kringle 5 specific Mab1-892-230 was diluted in 20 mM PBS, pH 7.4, to a final concentration of1.25 mg/mL. One-hundred microliters of the solution were added to eachwell of Maxisorb Nunc Immuno plates and incubated at room temperaturewith gentle shaking for two hours. The plates were washed five timeswith PBS-Tween and then blocked with 200 mL/well of 10% (v/v) FBS in 20mM PBS by incubation at 37° C. for one hour. The plates were stored at4° C. with plastic sealers. Before use, the plates were washed fivetimes with PBS-Tween.

[0105] b. Lp(a) Standard Curves: Lp(a) standards were prepared asdescribed in Example 3 above. Calibrators having 0, 0.0195, 0.039,0.078, 0.156, 0.312, 0.624, 1.248 and 2.5 mg/mL were prepared by serialdilution of the 2.5 mg/mL solution made from a Lp(a) standard in 3%(v/v) FBS in 20 mM PBS at pH 7.4. One-hundred microliters of Lp(a)standards (in duplicate) were incubated in the anti-kringle 5 coatedplates, prepared as described above, at 37° C. for one hour. Afterwashing the plates five times with PBS-Tween, 100 mL of anti-kringle 4Mab 4D2-HRPO conjugate (prepared as described in Section 3c above) at0.25 mg/mL in 3% FBS in PBS was added to each well and incubated at 37°C. for one hour. The plates were washed with PBS-Tween eight times. Theenzyme substrate o-phenylenediamine (OPD) (100 mL of a standard solutionprepared from one OPD tablet/10 mL citrate buffer, pH 6; bothcommercially available from Abbott Laboratories, Abbott Park, Ill.) wasadded to the wells. After incubation for 5 minutes, the color reactionwas stopped with (100 mL of 1 N sulfuric acid. The plates were read at490 nm on a microplate reader (Bio-Tek). The Lp(a) concentrations weremultiplied by 400 to generate the standard curves because the plasmasamples would be diluted 400 fold prior to performing the assay. A plotof Lp(a) concentration versus absorbance was prepared from the resultingdata. FIG. 10 is illustrative of such a plot.

[0106] c. Lp(a) Immunoassay: Plasma samples were diluted 400-fold with3% w/v FBS in 20 mM PBS, at pH 7.4. One-hundred microliters of thediluted samples were added to each well of the kringle 5 coated platesand the plates were incubated at 37° C. for one hour. After washing theplates five times with PBS-Tween, 100 mL of anti-kringle 4 Mab 4D2-HRPOconjugate (0.25 mg/mL in 3% w/v FBS in 20 mM PBS, pH 7.4) were added toeach well. The plates were incubated at 37° C. for one hour and thenwashed ten times with PBS-Tween. One-hundred microliters of a freshlyprepared solution of o-phenylenediamine in citrate buffer (prepared asabove) were added to each well and after five minutes the reaction wasquenched with 100 mL of 1 N sulfuric acid. The absorbance of each wellwas measured on a Bio-Tek microplate reader at 490 nm. TheLp(a)-cholesterol concentration for each sample was then determined froma standard curve of absorbance versus Lp(a)-cholesterol concentration,prepared as described above. The calibrators and the plasma samples wereassayed on the same plate to minimize the effect of variations in thereagents, materials or conditions.

[0107] The Lp(a) concentrations of the samples determined using a TERUMOELISA for Lp(a) (TERUMO Medical Corp., Elkton, Md.) and the methoddescribed above are shown in Table 2. The results in Table 2 illustratethe excellent correlation between TERUMO ELISA method and the presentmethod for determining Lp(a) levels in normal subjects (FIG. 9A)[correlation coefficient (r)=0.983; slope=0.898; intercept=1.85). Theresults obtained by the present method showed an excellent correlationwith the TERUMO ELISA method (r=0.953, intercept=0.18) but the slope was1.29 (FIG. 9B) on cardiac and diabetic patients. However, the TERUMOELISA tended to produce erroneous results for the cardiac and diabeticpatients, especially those with Lp(a) concentrations >50 mg/dL. Thelower Lp(a) values seen using the TERUMO ELISA method were notsurprising because of the assay's upper limit of 80 mg/dL Lp(a) and theslope of the standard curve above 50 mg/dL Lp(a).

EXAMPLE 5 Lp(a) Immunoassay with Anti-Kringle 5 Mab as Capture andHRPO-Labeled Anti-Apo(a) Pab for Detection

[0108] The preparation of the anti-kringle 5 plates, the generation ofthe Lp(a) standard curves and the Lp(a) immunoassay were performedexactly in the same way as described in Example 4 except thatHRPO-labeled anti-apo(a) Pab conjugate was used instead ofHRPO-anti-kringle 4 conjugate. The concentration of HRPO-anti-apo(a)conjugate, prepared as described in Section 3c, used was 1 mg/mL.

[0109] A typical standard curve is shown in FIG. 10. The results of theLp(a) assay are presented in Table 2 and the correlation between theLp(a) concentrations measured by the TERUMO ELISA and the present formatare shown in FIGS. 11A and 11B. Excellent correlation between the TERUMOELISA method and the present method was obtained in normal subjects(FIG. 11A) [r=0.95; slope=2.80; intercept=1.00]. On the other hand, thecorrelation between the two methods in cardiac patients and diabeticsubjects (FIG. 11B) showed results similar to those observed in Example4 above. The correlation between these methods in the patient populationwere: correlation coefficient (r)=0.95; intercept=1.54; slope=1.31. Theobserved high slope in this format is attributed to explanations similarto those discussed in Example 4c above.

EXAMPLE 6 Lp(a) Immunoassay with Anti-Kringle 4 Mab as Capture andHRPO-Labeled Anti-Kringle 5 Mab for Detection

[0110] a. Anti-kringle 4 Coated Plates: The kringle 4 specific Mab 4D2previously described was diluted in 20 mM PBS, pH 7.4, to a finalconcentration of 2.5 mg/mL. One-hundred microliters of the solution wereadded to each well of Maxisorb Nunc Immuno plates and processed asdescribed in Example 4a above.

[0111] b. Lp(a) Standard Curves: Lp(a) standards were prepared asdescribed in Example 3 above. Calibrators having 0, 0.0195, 0.039,0.078, 0.156, 0.312, 0.624, 1.248 and 2.5 mg/mL were prepared by serialdilution of the 2.5 mg/mL solution made from a Lp(a) standard in 3%(v/v) FBS in 20 mM PBS at pH 7.4. One-hundred microliters of Lp(a)standards (in duplicate) were incubated in the anti-kringle 4 coatedplates described above at 37° C. for one hour. After washing the platesfive times with PBS-Tween, 100 mL of anti-kringle 5 Mab-HRPO conjugate(prepared as described in Section 3c above) at 0.25 mg/mL in 3% FBS inPBS was added to each well and incubated at 37° C. for one hour. Theremainder of the procedure was exactly the same as described in Example4c except that the color development was stopped with 1 N sulfuric acidat 10 minutes. The Lp(a) concentrations were multiplied by 100 togenerate the standard curves because the plasma samples would be diluted100 fold prior to performing the assay. A plot of Lp(a) concentrationversus absorbance was prepared from the resulting data. FIG. 12 isillustrative of such a plot.

[0112] c. Lp(a) Immunoassay: Plasma samples were diluted 100-fold with3% w/v FBS in 20 mM PBS, at pH 7.4. One-hundred microliters of thediluted samples were added to each well of the kringle 4 coated platesand the plates were incubated at 37° C. for one hour. After washing theplates five times with PBS-Tween, 100 mL of anti-kringle 5 Mab HRPOconjugate (2 mg/mL in 3% w/v FBS in 20 mM PBS at pH 7.4) were added toeach well. The plates were incubated at 37° C. for one hour. Theremainder of the procedure was as described in Example 4c above exceptthe color development with the substrate was for 10 minutes. Thecalibrators and the plasma samples were assayed on the same plate tominimize the effect of variations in the reagents, materials orconditions.

[0113] The Lp(a) concentrations of the samples are shown in Table 2 andthe correlations between the Lp(a) concentrations measured by the TERUMOELISA method and the present format are shown in FIGS. 13A and 13B. Thecorrelation between the methods in normal subjects showed morescattering than the two other formats described in Examples 4 and 5. Thecorrelation between the two methods in normal subjects (FIG. 13A) had acorrelation coefficient (r)=0.91; intercept=2.81; slope=0.938. Thecorrelation between the two methods in patients is reasonable below anLp(a) concentration of 50 mg/dL (FIG. 13B); the correlations werer=0.924; intercept=−1.20; slope=1.07. The results with the presentformat indicate that the binding of labeled anti-kringle 5 Mab to theLp(a) captured on the immobilized anti-kringle 4 Mab is comparativelyweak. The results can possibly be improved by increasing theconcentration of the labeled conjugate and/or incubation time.

EXAMPLE 7 Lp(a) Immunoassay with Anti-Apo(a) as Capture and HRPO-LabeledAnti-Kringle 5 Mab for Detection

[0114] a. Anti-Apo(a) Coated Plates: The sheep anti-apo(a) polyclonalantibody previously described was diluted in 20 mM PBS, pH 7.4, to afinal concentration of 2.5 mg/mL. One-hundred microliters of thesolution were added to each well of Maxisorb Nunc Immunoplates andprocessed as described in Example 4a above.

[0115] b. Standard Curves and Lp(a) Immunoassay: The generation of theLp(a) standard curves and the Lp(a) immunoassay were performed asdescribed in Examples 6a and 6b. The standard curve with this format isillustrated in FIG. 14. The correlations between the Lp(a)concentrations as measured by the TERUMO ELISA method and the presentformat in normal subjects and in patients are shown in FIGS. 15A and15B, respectively. The correlations were: normal subjects r=0.91;intercept=0.80; slope=0.798; patients r=0.96; intercept=0.994;slope=0.734. The results indicate a similar situation to that seen inExample 6. It may be possible to improve the results by increasing theconcentration of the labeled anti-kringle 5 conjugate, altering theconcentration of the capture anti-apo(a) polyclonal antibody on thesolid phase and/or the incubation time.

EXAMPLE 8 Lp(a)-Cholesterol Assay with Anti-Kringle 5 Mab

[0116] a. Anti-kringle 5 Coated Plates: The kringle 5 specific Mab1-892-230 was diluted in 20 mM PBS, pH 7.4 to a final concentration of 5mg/mL. One-hundred microliters of the solution were added to each wellof Maxisorb Nunc Immuno plates and incubated at room temperature withgentle shaking for two hours. The plates were washed five times withPBS-Tween and then blocked with 200 mL/well of 5% (v/v) BSA in 20 mM PBSby incubation at 37° C. for one hour. The plates were stored at 4° C.with plastic sealers. Before use, the plates were washed five times withPBS-Tween.

[0117] b. Lp(a)-Cholesterol Standard Curves: Lp(a)-cholesterol standardswere prepared from Lp(a) standard solutions as described in Example 3.Calibrators having Lp(a)-cholesterol concentrations of 0, 0.0213,0.0426, 0.0852, 0.170, 0.341, 0.682, 1.364 and 2.73 mg/mL were preparedby serial dilution of the 2.73 mg/mL solution made from a Lp(a) standardin 1% (w/v) alkali-treated casein in 20 mM PBS, pH 7.4. One-hundredmicroliters of Lp(A)-cholesterol standards (in duplicate) were incubatedin the anti-kringle 5 Mab coated plates prepared above, at 37° C. forone hour. After washing the plates five times with PBS Tween, 100 mL ofHRPO-digitonin conjugate (prepared as described in Section 3b above) at1 mg/mL in 1% alkali-treated casein in 20 mM PBS, pH 7.4, was added toeach well and incubated at 37° C. for 1 hour. The plates were thenprocessed as described in Example 4c. The concentrations ofLp(a)-cholesterol were multiplied by 100 to generate the calibrationcurve because the plasma samples would be diluted 100-fold prior toperforming the assay. A plot of Lp(a)-cholesterol concentration versusabsorbance was prepared from the resulting data. FIG. 16 is illustrativeof such a plot. Generally the calibrators and the plasma samples wereassayed on the same plate to minimize the effect of variations in thereagents, materials or conditions. The number and concentration ofcalibrators used can be readily altered depending on the desiredaccuracy of the results.

[0118] c. Lp(a)-Cholesterol Immunoassay: Plasma samples were diluted100-fold with 1% w/v alkali-treated casein in 20 mM PBS, pH 7.4.One-hundred microliters of the diluted samples were added to each wellof the kringle 5 Mab coated plates, and the plates were incubated at 37°C. for one hour. After washing the plates five times with PBS-Tween, 100mL of digitonin-HRPO conjugate (1 mg/mL in 1% w/v alkali-treated caseinin 20 mM PBS, pH 7.4) were added to each well and incubated at 37° C.for one hour. The remainder of the procedure is as described in Example4c.

[0119] The Lp(a)-cholesterol concentrations of the samples derived fromthe TERUMO ELISA method were calculated by multiplying the Lp(a)concentration by 0.3. The results of Lp(a)-cholesterol concentrationsusing the immunoassay method above are shown in Table 3. FIG. 17 showsthe correlation between the calculated Lp(a)-cholesterol values obtainedfrom the TERUMO ELISA Lp(a) concentrations and the direct immunoassaymethod of this invention. The correlations between the methods withnormal subjects are: r=0.967; intercept=1.51; slope=0.893.

[0120] The embodiments described and the alternative embodimentspresented are intended as examples rather than as limitations. Thus, thedescription of the invention is not intended to limit the invention tothe particular embodiments disclosed, but it is intended to encompassall equivalents and subject matter within the spirit and scope of theinvention as described above and as set forth in the following claims.TABLE I UPID PROFILES OF PLASMA SAMPLES Sample Sample Total-C HDL-C TrigLDL-FE LDL-UC Lp(a) No. ID mg/dL mg/dL mgldL mg/dL mg/dL mg/dL  1 CP 22044 201 136 144 2.5  2 SF 154 39 43 106 114 0.3  3 CF 227 38 80 161 15061.6  4 NS 177 38 104 118 119 6.2  5 W 244 37 108 185 172 78.8  6 RB 17337 94 117 119 44.9  7 LL 172 78 51 84 91 4.4  8 MP 162 46 71 101 99 33.5 9 AJ 186 43 178 108 124 4.1 10 PB 163 56 51 96 91 46 11 AY 275 50 114202 174 24.5 12 GO 282 43 180 203 196 2 13 LA 199 65 249 84 102 17.9 14MM 210 57 115 131 123 37 15 RN 217 41 127 ISO 156 27 16 BC 222 64 77 142134 10.3 17 RC 181 52 60 117 116 4.8 18 OL 148 52 74 81 76 13 19 JS 16244 71 104 98 1.4 20 MS 208 38 131 143 148 4 21 LT 182 70 37 104 104 1.522 JC 160 55 104 84 84 87 23 HB 155 34 68 107 107 1 24 TS 130 33 73 8388 0.97 25 RR 214 42 52 162 164 0.2 26 ML 236 61 108 154 145 4.84 27 EB163 65 44 89 89 1.7 28 JR 142 41 130 75 85 30 29 DK 166 35 82 115 101 230 GC 241 61 74 165 165 80 31 LR 135 43 77 77 68 7.4 32 SD 227 32 365122 137 1.2 33 JM08810 n/a n/a n/a n/a n/a 12 34 JM08806 n/a n/a n/a n/an/a 20 35 JM08657 211 40 99 150 147 33 36 JM08632 178 35 70 87 n/a 62.637 WO 197 55 44 133 123 42 38 5K 230 47 109 150 161 25.8 39 BP 215 63 68138 148 19.5 40 ES639 160 32 172 94 93 9.5 41 ES629 294 70 134 197 20514.4 42 ES21I 172 68 73 89 20? 0.3 43 ES600 289 36 299 193 202 2.3 44ES337 192 41 118 127 129 1.8 45 ESI61 176 54 68 108 111 22 46 ES147 24343 182 164 176 63 47 ES596 256 43 179 177 167 34 48 ES284 198 33 184 128113 60 49 ES652 157 66 156 60 71 2 50 ES651 228 70 91 140 144 75 51ES290 196 42 161 122 116 4.4 52 ES15 255 39 206 175 171 20 53 ES13 17335 93 119 115 14.7 54 ES572 345 50 313 232 234 28.3 55 ES129 221 59 146133 132 75 56 ES146 199 48 93 132 139 32 57 ES453 298 74 116 201 204 2.558 ES593 174 47 126 102 105 13.6 59 ES571 209 42 193 128 128 4.4 60ES151 238 40 214 155 163 20 61 ES2 172 45 120 103 105 10.5 62 ES17 25967 101 172 181 11 63 ES10 204 33 148 141 143 7.8 64 ES9 165 35 83 113120 1 65 ES19 194 48 131 120 125 53 66 ES12 225 35 151 160 167 50 67ES193 190 41 87 132 139 26 68 ES153 218 40 178 142 148 78 69 ES7 365 44411 — 225 27.5 70 ES651 228 70 91 140 144 75 71 ES11 249 41 185 171 1673.6 72 ES8 336 62 151 244 251 9.2 73 ES4 267 30 331 171 172 4.4 74 ES327200 52 170 114 104 52 75 E3 228 52 120 152 161 59.6 76 ES582 237 48 90171 169 30 77 ES14 246 55 101 171 167 7.6 78 ES127 309 39 68 256 266 8.979 D7 220 43 145 149 1.9 80 D7944 232 44 335 122 24.4 81 D746 348 40 350119 06.2 82 D1782 170 30 159 108 1.4 83 D2237 205 44 III 139 5 84 D1767204 51 136 126 71 85 D2713 183 59 91 106 13.3 86 D2892 312 46 481 1702.6 87 D1784 197 34 193 124 4.6 88 D2714 240 38 278 147 66.2 89 D2165250 36 541 106 0.6 90 D2429 169 32 107 116 3.1 91 D2166 211 56 319 918.1 92 D2283 303 18 394 206 4.3 93 D2242 200 52 150 118 8 94 D1968 24947 175 167 0.6 95 D1765 316 38 294 219 66.2 96 D2465 164 28 250 85 11.897 D2743 164 41 108 102 26.9 98 D2688 197 35 191 124 32 99 D1983 136 41229 49 25.4 100  D1494 167 28 240 91 0.9 101  D1619 232 61 69 157 17.2102  D2298 129 37 109 70 6.3 103  D2294 215 48 281 112 14.7 104  D1766255 38 200 177 10.9 105  D2426 185 46 215 96 1.9 106  D1517 195 40 44366 2.93 107  D2310 136 37 134 72 0.4 108  D7986 213 46 245 118 51.8 109 D25 193 36 121 133 131 51.3 110  D26 214 48 75 151 161 1.7 111  D27 19940 142 131 18.8 112  D28 106 46 60 48 57 7.87 113  D29 166 38 30 98 10724.3 114  D30 227 55 127 147 174 N/D 115  D34 164 72 58 81 77 N/D 116 D35 221 74 101 127 118 N/D

[0121] TABLE 2 Lp(a) CONCENTRATION BY IMMUNOASSAY Lp(a) Lp(a) Lp(a)Lp(a) Lp(a) mg/dL mg/dL mg/dL mg/dL Sample Sample mg/dL Format FormatFormat Format No. ID* Ref Meth** 1 2 3 4 1 CP 2.5 3 2.6 4 1 2 SF 0.3 0.20.2 1 0.3 3 CF 61.6 58 70 48 50 4 NS 6.2 8 9 13 6 5 DW 78.8 71 70 59 406 RB 44.9 33.5 35 46 43 7 LL 4.4 4 5.8 3.5 3 8 MP 33.5 35 60 67 56 9 AJ4.1 3.5 4.9 2 2 10 PB 46 48 59 73 66 11 AY 24.5 33 30 28 20 12 GO 2 1.51.5 1.4 0.6 13 LA 17.9 31 31 18 15 14 MM 37 41 47 66 49 15 RN 27 25 4127 19 16 BC 10.3 14 12.6 12.8 5 17 RC 4.8 6.5 5.8 4.7 2.2 18 OL 13 13.812.6 13 5 19 JS 1.4 1 1 1.2 0.5 20 MS 4 5.3 6 3.2 3 21 LT 1.5 0.5 0.50.2 0.3 22 JC 87 77 70 56 62 23 HB 1 1 1 0.5 0.4 24 TS 0.97 0.6 0.5 0.80.3 25 RR 0.2 0.1 0.1 0.1 0.1 26 ML 4.84 6 6.5 3 2.4 27 EB 1.7 1 1 0.50.4 28 JR 30 33 32 26 26 29 DK 2 2 2 1 0.5 30 GC 80 67 89 88 62 31 LR7.4 9.8 10.7 5 4 32 SD 1.2 0.5 0.6 2 0.2 33 JMO8810 12 15 22 19 14 34JM08806 20 17 24 23 15 35 JM08657 33 24 24 38 20 36 JM08632 62.6 62 7669 50 37 WO 42 43 39.5 43.5 42 38 SK 25.8 28 38 25 13 39 BP 19.5 18 2421 16 40 ES639 9.5 8.2 6.7 8 2.5 41 ES629 14.4 16.5 22 15 8.7 42 ES2110.3 0.5 0.8 0.8 0.8 43 ES600 2.3 3.6 6.1 2.3 2 44 ES337 1.8 1 1.2 1.50.5 45 ES161 22 24.5 36 27 24 46 ES147 63 83 88 59 48 47 ES596 34 46 5041 41 48 ES284 60 62.3 65 66 48 49 ES652 2 1.4 1.6 2.1 1 50 ES651 75 8183 83 49 51 ES290 4.4 4.7 7 3.1 3.5 52 ES15 20 18.5 25.8 18.5 16 53 ES1314.7 18.4 24.4 17.1 17 54 ES572 28.3 35 44 27.4 35 55 ES129 75 65 64 6549.5 56 ES146 32 40 50 27 22 57 ES453 2.5 4.5 6.4 1.7 1.6 58 ES593 13.616.5 20.7 15 13 59 ES571 4.4 4.1 6.4 3 3.5 60 ES151 20 27 35 31 23 61ES2 10.5 14 26 7.7 7.2 62 ES17 11 12 12 12 11 63 ES10 7.8 6 5.4 8.5 4.264 ES9 1 0.5 0.6 1 0.5 65 ES19 53 69.2 82 64 42 66 ES12 50 75 75 65 4667 ES193 26 46 44 29 28 68 ES153 40 35 38 44 34 69 ES7 27.5 24.34 34.835 22 70 ES651 75 81 92 83 49 71 ES11 3.6 6 2.1 4 4.2 72 ES8 9.2 14 29.67 6 73 ES4 4.4 3 5 2.4 2.2 74 ES327 52 85 79 45 47 75 ES3 59.6 55.4 64.541 26 76 ES582 17 21.5 22 14 12.5 77 ES14 7.6 14 10 14 14 78 ES127 8.911 12.5 6.5 6 79 D7 1.9 1.7 1.9 1.3 1.1 80 D7944 24.4 36.1 38.7 43.523.7 81 D746 6.2 7.1 6 2.8 3 82 D1782 1.4 1.3 1.5 0.4 1 83 D2237 5 5.45.4 2 2.9 84 D1767 71 115 100 115 48 85 D2713 13.3 22.3 19.7 8.6 9.1 86D2892 2.6 3.7 3.6 1.4 1.6 87 D1784 4.6 5.2 4.7 1.5 2.1 88 D2714 66.2 104100 45 46 89 D2165 0.6 0.7 0.7 0.2 0.3 90 D2429 3.1 4.3 4.8 1.6 2.7 91D2166 8.1 12.3 12.6 6.7 7.4 92 D2283 4.3 6.1 7.2 3.1 4 93 D2242 8 8.9 85.7 3.1 94 D1968 0.6 0.2 0.2 0.2 0.2 95 D1765 66.2 107 110 110 55.2 96D2465 11.8 19.3 20.7 20 12.8 97 D2743 26.9 36 29 13.5 13.8 98 D2688 3253 52 33 30 99 D1983 25.4 38 42 22.4 21 100 D1494 0.9 0.5 0.7 0.2 0.2101 D1619 17.2 29.6 28.6 12 13.3 102 D2298 6.3 9.5 11.5 5 5.3 103 D229414.7 18 16.4 12.8 6.3 104 D1766 10.9 16.4 18 8.1 8.9 105 D2426 1.9 1.11.3 0.6 0.5 106 D1517 2.93 6.9 6.9 2.2 2.4 107 D2310 0.4 0.2 0.3 0.1 0.1108 D7986 51.8 55 69.4 32 46.2 109 D25 51.3 92.4 94.9 69.7 44.7 110 D261.7 1 1.3 0.8 0.5 111 D27 18.8 25.6 25.7 19.6 16.5 112 D28 7.87 7.4 7.34.7 3.9 113 D29 24.3 29.7 30 19.4 15.8 114 D30 55 63 65 29 33.8 115 D341.7 1 1.6 1 0.6 116 D35 58 101 100 50 45.5

[0122] TABLE 3 Lp(a)-CHOLESTEROL BY IMMUNOASSAY Lp(a) Lp(a)-C mg/dLLp(a)-C** mg/dL Sample No. Sample ID Ref Meth** mg/dL EIA  1 CP 2.5 0.81.5  2 SF 0.3 0.1 0.1  3 CF 61.6 18.5 18.0  4 NS 6.2 1.9 5.0  5 DW 78.823.6 19.0  6 RB 44.9 13.5 13.0  7 LL 4.4 1.3 2.9  8 MP 33.5 10.1 10.6  9AJ 4.1 1.2 5.0 10 PB 46 13.8 11.6 11 AY 24.5 7.4 7.2 12 GO 2 0.6 0.7 13LA 17.9 5.4 7.3 14 MM 37 11.1 10.1 15 RN 27 8.1 8.0 16 BC 10.3 3.1 5.017 RC 4.8 1.4 8.5 18 OL 13 3.9 6.6 19 JS 1.4 0.4 2.2 20 MS 4 1.2 4.9 21LT 1.5 0.5 0.1 22 JC 87 26.1 25.6 23 HB 1 0.3 0.6 24 TS 0.97 0.3 0.6 25RR 0.2 0.1 0.1 26 ML 4.84 1.5 4.0 27 EB 1.7 0.5 0.1 28 JR 30 9.0 10.3 29DK 2 0.6 3.7 30 GC 80 24.0 26.0 31 LR 7.4 2.2 2.2 32 SD 1.2 0.4 0.1 33JMO8810 12 3.6 6.0 34 JMO8806 20 6.0 6.0 35 JMO8657 33 9.9 9.7 36JMO8632 62.6 18.8 20.0 37 WO 42 12.6 13.0 38 DG 0.8 0.2 0.7 39 SK 19.65.9 5.9 40 ES639 9.5 2.9 7.6 41 ES629 14.4 4.3 13.8 42 ES211 0.3 0.1 2.043 ES600 2.3 0.7 9.2 44 ES337 1.8 0.5 1.3 45 ES161 22 6.6 6.8 46 ES14763 18.9 12.5 47 ES596 34 10.2 11.3 48 ES284 60 18.0 21.3 49 ES652 2 0.61.9 50 ES651 75 22.5 17.6 51 ES290 4.4 1.3 2.7 52 ES15 20 6.0 10.5 53ES13 14.7 4.4 8.5 54 ES572 28.3 8.5 10.3 55 ES129 75 22.5 22.5 56 ES14632 9.6 17.6 57 ES453 2.5 0.8 6.8 58 ES593 13.6 4.1 10.1 59 ES571 4.4 1.34.8 60 ES151 20 6.0 8.0 61 ES2 10.5 3.2 6.4 62 ES17 11 3.3 15.1 63 ES107.8 2.3 13.4 64 ES9 1 0.3 0.5 65 ES19 53 15.9 21.3 66 ES12 50 15.0 21.667 ES193 26 7.8 17.3 68 ES153 40 12.0 22.8 69 ES7 27.5 8.3 19.0 70 ES65175 22.5 19.5 71 ES11 3.6 1.1 4.9 72 ES8 9.2 2.8 18.4 73 ES4 4.4 1.3 3.674 ES327 52 15.6 24.7 75 ES3 59.6 17.9 17.8 76 ES582 17 5.1 11.0 77 ES147.6 2.3 17.5 78 ES127 8.9 2.7 8.0

[0123]

What is claimed is:
 1. A method for determining the amount of Lp(a) in asample comprising the steps of: (a) contacting said sample and an Lp(a)specific binding agent coupled to a solid support wherein said Lp(a)specific binding agent is a monoclonal antibody or fragment thereof thatspecifically binds to kringle 5 of apo(a) for a time and underconditions to form binding agent-Lp(a) complexes; and (b) determiningthe amount of Lp(a) bound to said binding agent-Lp(a) complexes.
 2. Themethod of claim 1 wherein said monoclonal antibody binds tosubstantially all Lp(a) via kringle 5, to plasminogen at less than 1% ofLp(a) binding and to LDL, VLDL, IDL, and HDL at less than 2% of Lp(a)binding.
 3. The method of claim 1 wherein said monoclonal antibody isselected from the group consisting of 1-532-266, 1-390-191, 1-458-165,1-892-230, 1-292-189, 1-431-378, 1-746-183, and 1-546-264.
 4. The methodof claim 2 wherein the solid support is selected from the groupconsisting of nitrocellulose, latex, nylon, polystyrene, beads,particles, magnetic particles, and glass fiber.
 5. The method of claim 1further comprising the step of separating said solid support from saidsample before determining the amount of Lp(a) bound to said bindingagent-Lp(a) complexes.
 6. The method of claim 1 further comprisingcontacting an indicator reagent with said sample and said Lp(a) specificbinding agent prior to step (b).
 7. The method of claim 6 wherein saidLp(a) specific binding agent binds to substantially all Lp(a) viakringle 5, to plasminogen at less than 1% of Lp(a) binding and to LDL,VLDL, IDL and HDL at less than 2% of Lp(a) binding.
 8. The method ofclaim 6 wherein said indicator reagent is selected from the groupconsisting of K4 specific monoclonal antibody, K4 polyclonal antibody,K4/K5 monoclonal antibody, K4/K5 polyclonal antibody and fragments ofeach.
 9. The method of claim 6 further comprising the step of separatingsaid solid support from said sample before determining the amount ofLp(a) bound to said binding agent-Lp(a) complexes.
 10. The method ofclaim 6 wherein said Lp(a) specific binding agent is selected from thegroup consisting of 1-532-266, 1-390-191, 1-458-165, 1-892-230,1-292-189, 1-431-378, 1-746-183, and 1-546-264.
 11. A method fordetermining the amount of Lp(a) in a sample comprising the steps of: (a)contacting said sample, an indicator reagent, and a capture reagentbound to a solid support wherein said indicator reagent is a labeledmonoclonal antibody or fragment thereof that specifically binds tokringle 5 of apo(a) for a time and under conditions to form capturereagent-Lp(a)-indicator reagent complexes; and (b) determining theamount of Lp(a) bound to said binding agent-Lp(a) complexes.
 12. Themethod of claim 11 wherein said indicator reagent binds to substantiallyall Lp(a) via kringle 5, to plasminogen at less than 1% of Lp(a) bindingand to LDL, VLDL, IDL and HDL at less than 2% of Lp(a) binding.
 13. Themethod of claim 11 wherein said capture reagent is selected from thegroup consisting of K4 specific monoclonal antibody or a fragmentthereof, K4 polyclonal antibody, K4/K5 monoclonal antibody, K4/K5polyclonal antibody and fragments of each.
 14. The method of claim 11further comprising the step of separating said solid support from saidsample before determining the amount of Lp(a) bound to said bindingagent-Lp(a) complexes.
 15. The method of claim 11 wherein said indicatorreagent is selected from the group consisting of 1-532-266, 1-390-191,1-458-165, 1-892-230, 1-292-189, 1-431-378, 1-746-183, and 1-546-264.16. A method for determining the amount of Lp(a) in a sample comprisingthe steps of: (a) contacting said sample, an Lp(a) specific bindingagent wherein said Lp(a) specific binding agent is conjugated to a firstcharged substance, and an indicator reagent wherein said indicatorreagent is monoclonal antibody or fragment thereof that specificallybinds to kringle 5 of apo(a) for a time and under conditions to formbinding agent-Lp(a)-indicator complexes; (c) contacting an insolublesolid phase material which is oppositely charged with respect to saidfirst charged substance, such that said solid phase material attractsand attaches to said first charged substance; and (d) determining theamount of Lp(a) bound to said binding agent-Lp(a)-indicator reagentcomplexes.
 17. The method of claim 16 wherein said monoclonal antibodyis selected from the group consisting of 1-532-266, 1-390-191,1-458-165, 1-892-230, 1-292-189, 1-431-378, 1-746-183, and 1-546-264.18. The method of claim 16 wherein said first charged substance is ananionic or cationic monomer or polymer.
 19. The method of claim 16wherein said indicator reagent binds to substantially all Lp(a) viakringle 5, to plasminogen at less than 1% of Lp(a) binding and to LDL,VLDL, IDL and HDL at less than 2% of Lp(a) binding.
 20. A method fordetermining the amount of Lp(a) in a sample comprising the steps of: (a)contacting said sample with an indicator reagent wherein said indicatorreagent is a monoclonal antibody or fragment thereof that specificallybinds to kringle 5 of apo(a) and with a solid support coated with Lp(a)for a time and under conditions to permit binding of said indicatorreagent with said Lp(a) in said test sample and with said bound Lp(a);and (b) determining said amount of Lp(a) in said test sample bydetecting the reduction in binding of said indicator reagent to saidsolid support as compared to the signal generated from a negative sampleto indicate the presence of Lp(a) in said test sample.
 21. The method ofclaim 20 wherein said monoclonal antibody binds to substantially allLp(a) via kringle 5, to plasminogen at less than 1% of Lp(a) binding andto LDL, VLDL, IDL and HDL at less than 2% of Lp(a) binding.
 22. Themethod of claim 20 wherein said monoclonal antibody is selected from thegroup consisting of 1-532-266, 1-390-191, 1-458-165, 1-892-230,1-292-189, 1-431-378, 1-746-183, and 1-546-264.
 23. The method of claim20 wherein at each occurrence therein, said indicator reagent isreplaced by labeled Lp(a) and said bound-Lp(a) is replaced by boundmonoclonal antibody or a fragment thereof that specifically binds tokringle 5 of apo(a).
 24. The method of claim 23 wherein said monoclonalantibody binds to substantially all Lp(a) via kringle 5, to plasminogenat less than 1% of Lp(a) binding and to LDL, VLDL, IDL and HDL at lessthan 2% of Lp(a) binding.
 25. The method of claim 23 wherein saidmonoclonal antibody is selected from the group consisting of 1-532-266,1-390-191, 1-458-165, 1-892-230, 1-292-189, 1-431-378, 1-746-183, and1-546-264.
 26. The method of claim 22 wherein at each occurrencetherein, said labeled Lp(a) is replaced by labeled kringle 5 of apo(a).27. A method for determining the amount of cholesterol associated withLp(a) in a sample comprising: (a) contacting a sample and a monoclonalantibody or fragment thereof that specifically binds to kringle 5 ofapo(a) wherein said antibody is coupled to a solid support; (b)separating said solid support from said sample; and (c) determining saidamount of cholesterol bound to said solid support.
 28. A monoclonalantibody specific for Lp(a) wherein said antibody binds to substantiallyall Lp(a) via kringle 5, to plasminogen at less than 1% of Lp(a) bindingand to LDL, VLDL, IDL and HDL at less than 2% of Lp(a) binding.
 29. Theantibody of claim 28 which is an IgG isotype.
 30. The antibody of claim29 selected from the group consisting of 1-532-266, 1-390-191, 1-458-165and 1-892-230.
 31. The antibody of claim 30 which is 1-892-230.
 32. Theantibody of claim 31 which is an IgM isotype.
 33. The antibody of claim32 selected from the group consisting of 1-292-189, 1-431-378,1-746-183, and 1-546-264.
 34. A monoclonal antibody specific for Lp(a)prepared by a method comprising the steps of: (a) immunizing a mouse ora rat with kringle 5 of apo(a) or a fragment thereof; (b) making asuspension of mouse or rat spleen cells; (c) fusing said spleen cellswith mouse or rat myeloma cells in the presence of a fusion promoter;(d) culturing said fused cells; (e) determining the presence ofanti-Lp(a) antibody in the culture media; (f) cloning a hybridomaproducing antibody that binds to substantially all Lp(a), to plasminogenat less than 1% of Lp(a) binding and to other lipoproteins, such as,LDL, VLDL, IDL and HDL at less than 2% of Lp(a) binding; and (g)obtaining said antibody from said hybridoma.
 35. A hybridoma cell linethat secretes a monoclonal antibody that binds to substantially allLp(a), to plasminogen at less than 1% of Lp(a) binding and to otherlipoproteins, such as, LDL, VLDL, IDL and HDL at less than 2% of Lp(a)binding.
 36. A hybridoma cell line that secretes a monoclonal antibodyselected from the group consisting of 1-532-266, 1-390-191, 1-458-165,1-892-230, 1-292-189, 1-431-378, 1-746-183, and 1-546-264.
 37. A testkit for the detection and quantification of Lp(a) in a plasma sample,comprising a reagent which specifically binds to kringle 5 of apo(a).38. The test kit of claim 37 wherein said reagent is labeled.